Methods and compositions relating to CD39-like polypeptides and nucleic acids

ABSTRACT

The present invention provides polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins. In particular, the polypeptides and polynucleotides of the invention comprise amino acid and nucleic acid sequences of novel CD39-like gene and gene products.

This application is a continuation application of U.S. application Ser.No. 09/240,639, now U.S. Pat. No. 6,350,447, filed on Jan. 29, 1999,entitled “Methods and Compositions Relating to CD-39-like Polypeptidesand Nucleic Acids,” which is herein incorporated by reference in itsentirety.

1. FIELD OF THE INVENTION

The present invention provides novel polynucleotides and proteinsencoded by such polynucleotides, along with therapeutic, diagnostic andresearch utilities for these polynucleotides and proteins.

2. BACKGROUND OF THE INVENTION

Technology aimed at the discovery of protein factors (including e.g.,cytokines, such as lymphokines, interferons, CSFs and interleukins) hasmatured rapidly over the past decade. The now routine hybridizationcloning and expression cloning techniques clone novel polynucleotides“directly” in the sense that they rely on information directly relatedto the discovered protein (i.e., partial DNA/amino acid sequence of theprotein in the case of hybridization cloning; activity of the protein inthe case of expression cloning). In addition, more recently, “indirect”cloning techniques have been utilized, such as signal sequence cloning,which isolates DNA sequences based on the presence of a nowwell-recognized secretory leader sequence motif, as well as variousPCR-based or low stringency hybridization cloning techniques, coupled incertain instances with database searching, have advanced the state ofthe art by making available large numbers of DNA/amino acid sequencesfor proteins that are known to have biological activity by virtue oftheir secreted nature in the case of leader sequence cloning, by virtueof the cell or tissue source in the case of PCR-based techniques and/orby virtue of their sequence similarity via database searches. It is tothese proteins and the polynucleotides encoding them that the presentinvention is directed.

3. SUMMARY OF THE INVENTION

The compositions of the present invention include novel isolatedpolypeptides, in particular, novel CD-39-like polypeptides, isolatedpolynucleotides encoding such polypeptides, including recombinant DNAmolecules, cloned genes or degenerate variants thereof, especiallynaturally occurring variants such as allelic variants, and antibodiesthat specifically recognize one or more epitopes present on suchpolypeptides.

The compositions of the present invention additionally include vectors,including expression vectors, containing the polynucleotides of theinvention, cells genetically engineered to contain such polynucleotidesand cells genetically engineered to express such polynucleotides.

The isolated polynucleotides of the invention include, but are notlimited to, a polynucleotide encoding a polypeptide comprising the aminoacid sequence of SEQ ID NO:2 (also referred to herein as “CD39L2”); or apolynucleotide encoding a polypeptide comprising amino acid residues72-93, 147-162, 191-211 OR 217-238 of SEQ ID NO:2.

In selected embodiments, such isolated polynucleotides of the inventionrepresents a polynucleotide comprising the nucleotide sequence of SEQ IDNO:1; or a polynucleotide comprising nucleotides 232-1599, 445-510,670-717, 802-864 or 880-945 of the nucleotide sequence of SEQ ID NO:1.

The polynucleotides of the present invention also include, but are notlimited to, a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:1 under highly stringent hybridizationconditions; a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:1 under moderately stringenthybridization conditions; a polynucleotide which is an allelic variantof any polynucleotide recited above; a polynucleotide which encodes aspecies homologue of any of the proteins recited above; or apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptide of SEQ ID NO:2.

The isolated polynucleotides of the invention still further include, butare not limited to, a polynucleotide encoding a polypeptide comprisingthe amino acid sequence of SEQ ID NO:4 (also referred to herein as“CD39L3”); or a polynucleotide encoding a polypeptide comprising aminoacid residues 55-76, 132-150, 177-199 or 213-234 of SEQ ID NO:4.

In selected embodiments, such isolated polynucleotides of the inventionrepresents a polynucleotide comprising the nucleotide sequence of SEQ IDNO:3; or a polynucleotide comprising nucleotides 83-1669, 245-310,476-532, 611-679 or 719-784 of the nucleotide sequence of SEQ ID NO:3.

The polynucleotides of the present invention also include, but are notlimited to, a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:3 under highly stringent hybridizationconditions; a polynucleotide that hybridizes to the complement of the1nucleotide sequence of SEQ ID NO:3 under moderately stringenthybridization conditions; a polynucleotide which is an allelic variantof any polynucleotide recited above; a polynucleotide which encodes aspecies homologue of any of the proteins recited above; or apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptide of SEQ ID NO:4.

The isolated polynucleotides of the invention still further include, butare not limited to, a polynucleotide encoding a polypeptide comprisingthe amino acid sequence of SEQ ID NO:6 (also referred to herein as“CD39L4”); or a polynucleotide encoding a polypeptide comprising aminoacid residues 47-68, 123-138, 167-187 or 193-214 of SEQ ID NO:6; or apolynucleotide encoding a polypeptide comprising the amino acid sequenceof SEQ ID NO:9 (also referred to herein as dCD39L4”); or apolynucleotide encoding amino acid residues 77-98, 153-167, 197-217 or223-242 of SEQ ID NO:9.

In one embodiment, such isolated polynucleotides of the inventionrepresents a polynucleotide comprising the nucleotide sequence of SEQ IDNO:5; or a polynucleotide comprising nucleotides 247-1530, 385-450,613-660, 745-807 or 823-888 of the nucleotide sequence of SEQ ID NO:5.

The polynucleotides of the present invention also include, but are notlimited to, a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:5 under highly stringent hybridizationconditions; a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:5 under moderately stringenthybridization conditions; a polynucleotide which is an allelic variantof any polynucleotide recited above; a polynucleotide which encodes aspecies homologue of any of the proteins recited above; or apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptide of SEQ ID NO:6.

The isolated polynucleotides of the invention further include, but arenot limited to, a polynucleotide encoding a polypeptide comprising theamino acid sequence of SEQ ID NO:8 (also referred to herein as “mCD39L4”or “mNTPase”); or a polynucleotide encoding amino acid residues 46-67,122-140, 166-187 or 194-213 of SEQ ID NO:8.

In selected embodiments, such isolated polynucleotides of the inventionrepresents a polynucleotide comprising the nucleotide sequence of SEQ IDNO:7; or a polynucleotide comprising nucleotides 205-1599, 340-395,568-624, 700-765 or 784-843 of the nucleotide sequence of SEQ ID NO:7.

The polynucleotides of the present invention also include, but are notlimited to, a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:7 under highly stringent hybridizationconditions; a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:7 under moderately stringenthybridization conditions; a polynucleotide which is an allelic variantof any polynucleotide recited above; a polynucleotide which encodes aspecies homologue of any of the proteins recited above; or apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptide of SEQ ID NO:8.

The polynucleotides of the invention additionally include the complementof any of the polynucleotides recited above.

The isolated polypeptides of the invention further include, but are notlimited to, a polypeptide comprising the amino acid sequence of SEQ IDNO:2; or a polypeptide comprising amino acid residues 72-93, 147-162,191-211 OR 217-238 of SEQ ID NO:2.

The isolated polypeptides of the invention still further include, butare not limited to, a polypeptide comprising the amino acid sequence ofSEQ ID NO:4; or a polypeptide comprising amino acid residues 55-76,132-150, 179-199 or 213-234 of SEQ ID NO:4.

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising the amino acid sequence of SEQ ID NO:6; ora polypeptide comprising amino acid residues 47-68, 123-138, 167-187 or193-214 of SEQ ID NO:6.

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising the amino acid sequence of SEQ ID NO:8; ora polypeptide comprising amino acid residues 46-67, 122-140, 166-187 or194-213 of SEQ ID NO:8.

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising the amino acid sequence of SEQ ID NO:9; ora polypeptide comprising amino acid residues 77-98, 153-167, 197-217 or223-242 of SEQ ID NO:9.

Preferred embodiments include polypeptides that represent is matureforms of the polypeptides of the invention.

Polypeptide compositions of the present invention may further comprisean acceptable carrier, such as a hydrophilic, e.g., pharmaceuticallyacceptable, carrier.

The invention also relates to methods for producing a polypeptidecomprising growing a culture of the cells of the invention in a suitableculture medium, and purifying the protein from the culture or from anextract of the cells.

Polynucleotides according to the invention have numerous applications ina variety of techniques known to those skilled in the art of molecularbiology. These techniques include use as hybridization probes, use asprimers for PCR, use for chromosome and gene mapping, use in therecombinant production of protein, and use in generation of anti-senseDNA or RNA, their chemical analogs and the like. For example, when theexpression of an mRNA is largely restricted to a particular cell ortissue type, polynucleotides of the invention can be used ashybridization probes to detect the presence of the particular cell ortissue mRNA in a sample using, e.g., in situ hybridization.

In other exemplary embodiments, the polynucleotides are used indiagnostics as expressed sequence tags for identifying expressed genesor, as well known in the art and exemplified by Vollrath et al., Science258:52-59 (1992), as expressed sequence tags for physical mapping of thehuman genome.

The polypeptides according to the invention can be used in a variety ofconventional procedures and methods that are currently applied to otherproteins. For example, the polypeptides of the invention can be used asmolecular weight markers, and as a food supplement. In addition, apolypeptide of the invention can be used to generate an antibody thatspecifically binds the polypeptide.

Methods are also provided for preventing, treating or ameliorating amedical condition which comprises administering to a mammalian subject atherapeutically effective amount of a composition comprising a proteinof the present invention and a pharmaceutically acceptable carrier.

The polypeptides and polynucleotides of the invention can be utilized,for example, as part of methods for modulating ecto-ATPase activity andfor identifying compounds that can be utilized as part of methods formodulating ecto-ATPase activity. Among the processes that can bemodulated via such methods are processes involved in cell adhesion,apoptosis, vesicular transport, signalling, including purinergic,synaptic and neurotransmitter signalling, and purine recycling. Thepolypeptides of the invention having ADPase activity are also useful asanticoagulants and for inhibiting platelet aggregation. The polypeptidesof the invention can further be utilized as anti-thrombotic agents,anti-tissue graft rejection agents, and/or as part of methods forregulating neurotransmission by ATP in smooth muscle, peripheral gangliaor the brain.

The methods of the present invention further relate to methods fordetecting the presence of the polynucleotides or polypeptides of theinvention in a sample. Such methods can, for example, be utilized aspart of prognostic and diagnostic evaluation of disorders as recitedabove and for the identification of subjects exhibiting a predispositionto such conditions. Furthermore, the invention provides methods forevaluating the efficacy of drugs, and monitoring the progress ofpatients, involved in clinical trials for the treatment of disorders asrecited above.

The invention also provides methods for the identification of compoundsthat modulate the expression of the polynucleotides and/or polypeptidesof the invention. Such methods can be utilized, for example, for theidentification of compounds that can ameliorate symptoms of disorders asrecited above. Such methods can include, but are not limited to, assaysfor identifying compounds and other substances that interact with (e.g.,bind to) the polypeptides of the invention.

The methods of the invention also include methods for the treatment ofdisorders as recited above which may involve the administration of suchcompounds to individuals exhibiting symptoms or tendencies related todisorders as recited above. In addition, the invention encompassesmethods for treating diseases or disorders as recited above byadministering compounds and other substances that modulate the overallactivity of the target gene products. Compounds and other substances caneffect such modulation either on the level of target gene expression ortarget protein activity.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, 1E, and 1F. Top line: Nucleotide sequence of SEQID NO: 7, referred to herein as mNTPase or mCD39L4; bottom line: aminoacid sequence of SEQ ID NO: 8, referred to herein as mNTPase or mCD39L4.

FIGS. 2A, 2B, and 2C. Amino acid alignments of the full mNTPase(mCD39L4) amino acid sequence (SEQ ID NO:8) and the most closely relatedother NTPase proteins: garden pea NTpase (SEQ ID NO:10), potato apyrase(SEQ ID NO:11), yeast GDPase (SEQ ID NO:12). Identical residues areindicated by double underlining, while conserved residues are indicatedby single underlining. Alignments were made with pileup and boxshadefrom the Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison Wis.

FIGS. 3A, 3B, 3C, and 3D. Alignment of 12 members of the NTPase (orCD39-like) gene family indicating the conserved apryase regions I-IV.CD39=human (from Accession No. S73813; SEQ ID NO:13), ratCD39=rat (fromAccession No. gi11754710; SEQ ID NO: 14), CD39L1=human (Accession No.U91510; SEQ ID NO:15), ChiATPase=chicken (from Accession No. U74467; SEQID NO:16), peaNTPase=garden pea (from Accession No. P52914; SEQ IDNO:10), potRROP1=potato RROP1 gene (from Accession No. gi11381633; SEQID NO:11), yGDA1+y71 KD=yeast genes (from Accession Nos.sp1P32621+sp1P40009; SEQ ID NO: 12), hCD39L2=CD39L2, celegans=C. Elegansgene (from Accession No. gi11086594; SEQ ID NO:17). Identical residuesare indicated by double underlining, conserved residues are indicated bysingle underlining. Alignments were made with pileup and boxshade fromthe Wisconsin Package 9.0, Genetics Computer Group (GCG), Madison, Wis.Conserved portions of ACRs I-IV are boxed.

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, and 4H. Top line: Nucleotide sequenceof SEQ ID NO:1, referred to herein as CD39L2; bottom line: amino acidsequence of SEQ ID NO:2, referred to herein as CD39L2.

FIGS. 5A, 5B, 5C, 5D, and 5E. Comparison of the hydrophobicitypredictions for the amino acid sequences of members of the humanCD39-like gene family. Predictions were made using the Topred-II 1.1program (Claros, M. G. & Von Hejine, G., 1994, Comput. Appl. Biosci.10:685-686; putative setting=0.5; certain setting=1.0).

FIGS. 6A, 6B, 6C, 6D, 6E, 6F, 6G, and 6H. Top line: Nucleotide sequenceof SEQ ID NO:3, referred to herein as CD39L3; bottom line: amino acidsequence of SEQ ID NO:4, referred to herein as CD39L3.

FIGS. 7A, 7B, 7C, 7D, 7E, and 7F. Top line: Nucleotide sequence of SEQID NO:5, referred to herein as CD39L4; bottom line: amino acid sequenceof SEQ ID NO:6, referred to herein as CD39L4.

FIGS. 8A, 8B, 8C, and 8D. Amino acid alignments of the full-lengthprotein sequences for human members of the CD39-like gene family. CD39(from Accession No. S73813; SEQ ID NO:13), CD39L1 (from Accession No.U91510; SEQ ID NO: 15), CD39L2 (it is noted that the CD39L2 polypeptideillustrated here depicts a derived amino acid sequence that is encodedfrom the ATG codon beginning at nucleotide 148 (see FIG. 4A) and,therefore, includes an additional 28 amino acid residues N-terminal tothose depicted in FIG. 4A; this form of CD39L2 is also intended to beincluded within the scope of the present invention), CD39L3, CD39L4.

Identical residues are indicated by double underlining and conservedresidues are indicated by single underlining. Spaces in the sequencesare indicated by a dash. Apyrase regions (ACRs) are indicated by arrows,with conserved portions of ACRs I-IV are highlighted by the boxedsections. Alignments were made using pileup and boxshade from theWisconsin Package Version 9.0 Genetics Computer Group (GCG), Madison,Wis.

FIGS. 9A, 9B, 9C, 9D, and 9E. Amino acid sequence of dCD39L4(“dNTPase”;SEQ ID NO:9) and alignment of the amino acid sequence with the mostclosely related members of the CD39-like gene family. peaGDP, garden peaNTPase (from Accession No. P52194; SEQ ID NO:10); ptoapyrase, potatoRROP1 gene (from Accession No. gi11381633; SEQ ID NO:11); CD39L2;CD39L4, and yGDPase, yeast yGDA1 gene (from Accession No. sp1P32621; SEQID NO:12). Apyrase regions (ACRs) are indicated by arrows, withconserved portions of ACRs I-IV are highlighted by the boxed sections.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Definitions

The term “nucleotide sequence” refers to a heteropolymer of nucleotidesor the sequence of these nucleotides. The terms “nucleic acid” and“polynucleotide” are also used interchangeably herein to refer to aheteropolymer of nucleotides. Generally, nucleic acid segments providedby this invention may be assembled from fragments of the genome andshort oligonucleotide linkers, or from a series of oligonucleotides, orfrom individual nucleotides, to provide a synthetic nucleic acid whichis capable of being expressed in a recombinant transcriptional unitcomprising regulatory elements derived from a microbial or viral operon,or a eukaryotic gene. In alternate embodiments, a nucleotide sequence,polynucleotide or nucleic acid can correspond to a genomic sequence (e ,can contain intron as well as exon sequence) or cDNA sequences (that is,contains no intron sequence).

The terms “oligonucleotide fragment” or a “polynucleotide fragment”,“portion,” or “segment” is a stretch of polypeptide nucleotide residueswhich is long enough to use in polymerase chain reaction (PCR) orvarious hybridization procedures to identify or amplify identical orrelated parts of mRNA or DNA molecules.

The terms “oligonucleotides” or “nucleic acid probes” are prepared basedon the polynucleotide sequences provided in the present invention.Oligonucleotides comprise portions of such a polynucleotide sequencehaving at least about 15 nucleotides and usually at least about 20nucleotides. Nucleic acid probes comprise portions of such apolynucleotide sequence having fewer nucleotides than about 6 kb,usually fewer than about 1 kb. After appropriate testing to eliminatefalse positives, these probes may, for example, be used to determinewhether specific mRNA molecules are present in a cell or tissue or toisolate similar nucleic acid sequences from chromosomal DNA as describedby Walsh et al. (Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).

The term “probes” includes naturally occurring or recombinant orchemically synthesized single- or double-stranded nucleic acids. Theymay be labeled by nick translation, Klenow fill-in reaction, PCR orother methods well known in the art. Probes of the present invention,their preparation and/or labeling are elaborated in Sambrook, J. et al.,1989, Molecular Cloning: A Laboratory Manual, Cold spring HarborLaboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols inMolecular Biology, John Wiley & Sons, New York N.Y., both of which areincorporated herein by reference in their entirety.

The “oligonucleotide fragment,” “polynucleotide fragment,” “portion,”“segment,”, “oligonucleotide” or “nucleic acid probe” is at least about15, and preferably at least about 50, 100, 200, 300, 400, 500, 600, 700or 800 nucleotides in length.

The term “stringent” is used to refer to conditions that are commonlyunderstood in the art as stringent. Stringent conditions can includehighly stringent conditions (i.e., hybridization to filter-bound DNAunder in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringentconditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.).

In instances wherein hybridization of deoxyoligonucleotides isconcerned, additional exemplary highly stringent hybridizationconditions include washing in 6×SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos).

The term “recombinant,” when used herein to refer to a polypeptide orprotein, means that a polypeptide or protein is derived from recombinant(e.g., microbial or mammalian) expression systems. “Microbial” refers torecombinant polypeptides or proteins made in bacterial or fungal (e.g.,yeast) expression systems. As a product, “recombinant microbial” definesa polypeptide or protein essentially free of native endogenoussubstances and unaccompanied by associated native glycosylation.Polypeptides or proteins expressed in most bacterial cultures, e.g., E.coli, will be free of glycosylation modifications; polypeptides orproteins expressed in yeast will have a glycosylation pattern in generaldifferent from those expressed in mammalian cells.

The term “recombinant expression vehicle or vector” refers to a plasmidor phage or virus or vector, for expressing a polypeptide from a DNA(RNA) sequence. An expression vehicle can comprise a transcriptionalunit comprising an assembly of (1) a genetic element or elements havinga regulatory role in gene expression, for example, promoters orenhancers, (2) a structural or coding sequence which is transcribed intomRNA and translated into protein, and (3) appropriate transcriptioninitiation-and termination sequences. Structural units intended for usein yeast or eukaryotic expression systems preferably include a leadersequence enabling extracellular secretion of translated protein by ahost cell. Alternatively, where recombinant protein is expressed withouta leader or transport sequence, it may include an N-terminal methionineresidue. This residue may or may not be subsequently cleaved from theexpressed recombinant protein to provide a final product.

The term “recombinant expression system” means host cells which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit extrachromosomally.Recombinant expression systems as defined herein will expressheterologous polypeptides or proteins upon induction of the regulatoryelements linked to the DNA segment or synthetic gene to be expressed.This term also means host cells which have stably integrated arecombinant genetic element or elements having a regulatory role in geneexpression, for example, promoters or enhancers. Recombinant expressionsystems as defined herein will express polypeptides or proteinsendoqenous to the cell upon induction of the regulatory elements linkedto the endogenous DNA segment or gene to be expressed. The cells can beprokaryotic or eukaryotic.

The term “open reading frame,” ORF, means a series of nucleotidetriplets coding for amino acids without any termination codons and is asequence translatable into protein.

The term “expression modulating fragment,” EMF, means a series ofnucleotides which modulates the expression of an operably linked ORF oranother EMF.

As used herein, a sequence is said to “modulate the expression of anoperably linked sequence” when the expression of the sequence is alteredby the presence of the EMP. EMFs include, but are not limited to,promoters, and promoter modulating sequences (inducible elements). Oneclass of EMFs are fragments which induce the expression or an operablylinked ORF in response to a specific regulatory factor or physiologicalevent.

As used herein, an “uptake modulating fragment,” UMF, means a series ofnucleotides which mediate the uptake of a linked DNA fragment into acell. UMFs can be readily identified using known UMFs as a targetsequence or target motif with the computer-based systems describedbelow.

The presence and activity of a UMF can be confirmed by attaching thesuspected UMF to a marker sequence. The resulting nucleic acid moleculeis then incubated with an appropriate host under appropriate conditionsand the uptake of the marker sequence is determined. As described above,a UMF will increase the frequency of uptake of a linked marker sequence.

The term “active” refers to those forms of the polypeptide which retainthe biological and/or immunological activities of any naturallyoccurring polypeptide.

The term “biologically active” refers to the biological activity of anaturally occurring polypeptide as well as to the ability of thepolypeptide to exhibit an immunological activity. A polypeptide exhibitsan “immunological activity” when antibodies can be generated that aredirected against the polypeptide.

The term “naturally occurring polypeptide” refers to polypeptidesproduced by cells that have not been genetically engineered andspecifically contemplates various polypeptides arising frompost-translational modifications of the polypeptide including, but notlimited to, acetylation, carboxylation, glycosylation, phosphorylation,lipidation and acylation.

The term “mature” refers to a polypeptide that has beenpostranslationally modified or that corresponds in primary amino acidsequence to a polypeptide that has been postranslationally modified. Amature polypeptide includes, but is not limited to, a polypeptide whichcomprises a primary amino acid sequence that has been processed from a“dpre-,” “pro-,” or “pre-pro” amino acid sequence; a polypeptide whichcomprises a primary amino acid sequence corresponding to that of apolypeptide that has been processed from a “pre-,” “pro-,” or “pre-pro”amino acid sequence; a polypeptide that has been post-translationallymodified via such modifications as, for example, acetylation,carboxylation, glycosylation, phosphorylation, lipidation and acylation.

The term “derivative” refers to polypeptides chemically modified by suchtechniques as ubiquitination, labeling (e.g., with radionuclides orvarious enzymes), pegylation (derivatization with polyethylene glycol)and insertion or substitution by chemical synthesis of amino acids suchas ornithine, which do not normally occur in human proteins.

The term “recombinant variant” refers to any polypeptide differing fromnaturally occurring polypeptides by amino acid insertions, deletions,and substitutions, created using recombinant DNA techniques. Guidance indetermining which amino acid residues may be replaced, added or deletedwithout abolishing activities of interest, such as cellular trafficking,may be found by comparing the sequence of the particular polypeptidewith that of homologous peptides and minimizing the number of amino acidsequence changes made in regions of high homology.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements. Aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

“Insertions” or “deletions” are typically in the range of about 1 to 5amino acids. The variation allowed may be experimentally determined bysystematically making insertions, deletions, or substitutions of aminoacids in a epolypeptide molecule using recombinant DNA techniques andassaying the resulting recombinant variants for activity.

Alternatively, where alteration of function is desired, insertions,deletions or non-conservative alterations can be engineered to producealtered polypeptides. Such alterations can, for example, alter one ormore of the biological functions or biochemical characteristics of thepolypeptides of the invention. For example, such alterations may changepolypeptide characteristics such as ligand-binding affinities,interchain affinities, or degradation/turnover rate. Further, suchalterations can be selected so as to generate polypeptides that arebetter suited for expression, scale up and the like in the host cellschosen for expression. For example, cysteine residues can be deleted orsubstituted with another amino acid residue in order to eliminatedisulfide bridges.

As used herein, “substantially equivalent” can refer both to nucleotideand amino acid sequences, for example a mutant sequence, that variesfrom a reference sequence by one or more substitutions, deletions, oradditions, the net effect of which does not result in an adversefunctional dissimilarity between the reference and subject sequences.Typically, such a substantially equivalent sequence varies from one ofthose listed herein by no more than about 20%, i.e., the number ofindividual residue substitutions, additions, and/or deletions in asubstantially equivalent sequence, as compared to the correspondingreference sequence, divided by the total number of residues in thesubstantially equivalent sequence is about 0.2 or less. Such a sequenceis said to have 80% sequence identity to the listed sequence. Such asubstantially equivalent sequence can be routinely identified byapplying the foregoing algorithm.

In one embodiment, a substantially equivalent, e.g., mutant, sequence ofthe invention varies from a listed sequence by no more than 10%, i.e.,the number of individual residue substitutions, additions, and/ordeletions in a substantially equivalent sequence, as compared to thecorresponding reference sequence, divided by the total number ofresidues in the substantially equivalent sequence is about 0.1 or less.Such a sequence is said to have 90% sequence identity to the listedsequence. Such a substantially equivalent sequence can be routinelyidentified by applying the foregoing algorithm.

In an alternate embodiment a substantially equivalent sequence of theinvention varies from a listed sequence by no more than by no more than5%, i.e., the number of individual residue substitutions, additions,and/or deletions in a substantially equivalent sequence, as compared tothe corresponding reference sequence, divided by the total number ofresidues in the substantially equivalent sequence is about 0.05 or less.Such a sequence is said to have 95% sequence identity to the listedsequence. Such a substantially equivalent sequence can be routinelyidentified by applying the foregoing algorithm.

In yet another alternate embodiment, a substantially equivalent sequenceof the invention varies from a listed sequences by no more than 2%,i.e., the number of individual residue substitutions, additions, and/ordeletions in a substantially equivalent sequence, as compared to thecorresponding reference sequence, divided by the total number ofresidues in the substantially equivalent sequence is about 0.02 or less.Such a sequence is said to have 98% sequence identity to the listedsequence. Such a substantially equivalent sequence can be routinelyidentified by applying the foregoing algorithm.

Substantially equivalent, e.g., mutant, amino acid sequences accordingto the invention generally have at least 95% sequence identity with alisted amino acid sequence, whereas substantially equivalent nucleotidesequence of the invention can have lower percent sequence identities,taking into account, for example, the redundancy or degeneracy of thegenetic code. For the purposes of the present invention, sequenceshaving substantially equivalent biological activity and substantiallyequivalent expression characteristics are considered substantiallyequivalent. In a preferred embodiment, for the purposes of determiningequivalence, truncation of the mature sequence (e.g., via a mutationwhich creates a spurious stop codon) are disregarded.

Nucleic acid sequences encoding such substantially equivalent sequences,e.g., sequences of the recited percent identities, can also routinely beisolated and identified via standard hybridization procedures well knownto those of skill in the art.

Where desired, an expression vector may be designed to contain a “signalor leader sequence” which will direct the polypeptide through themembrane of a cell. Such a sequence may be naturally present on thepolypeptides of the present invention or provided from heterologousprotein sources by recombinant DNA techniques.

A polypeptide “fragment,” “portion,” or “segment” is a stretch of aminoacid residues of at least about 5 amino acids, often at least about 7amino acids or about at least about 9 to 13 amino acids, and, in variousembodiments, at least about 17, 25, 50, 75, 100, 150, 200, 300, 400 ormore amino acids. To be “active,” any polypeptide must have sufficientlength to display biologic and/or immunologic activity.

Recombinant variants encoding these same or similar polypeptides may besynthesized or selected by making use of the “redundancy” in the geneticcode. Various codon substitutions, such as the silent changes whichproduce various restriction sites, may be introduced to optimize cloninginto a plasmid or viral vector or expression in a particular prokaryoticor eukaryotic system. Mutations in the polynucleotide sequence may bereflected in the polypeptide or domains of other peptides added to thepolypeptide to modify the properties of any part of the polypeptide, tochange characteristics such as ligand-binding affinities, interchainaffinities, or degradation/turnover rate. Such variant nucleic acids andpolypeptides are to be considered part of the present invention.

The term “activated” cells as used herein refers to those cells that areengaged in extracellular or intracellular membrane trafficking,including the export of neurosecretory or enzymatic molecules as part ofa normal or disease process.

The term “purified” as used herein denotes that the indicated nucleicacid or polypeptide is present in the substantial absence of otherbiological macromolecules, e.g., polynucleotides, proteins, and thelike. In one embodiment, the polynucleotide or polypeptide is purifiedsuch that it constitutes at least 95% by weight, more preferably atleast 99.8% by weight, of the indicated biological macromoleculespresent (but water, buffers, and other small molecules, especiallymolecules having a molecular weight of less than 1000 daltons, can bepresent).

The term “isolated” as used herein refers to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) present with the nucleic acid or polypeptide in itsnatural source. In one embodiment, the nucleic acid or polypeptide isfound in the presence of (if anything) only a solvent, buffer, ion, orother component normally present in a solution of the same. The terms“isolated” and “purified” do not encompass nucleic acids or polypeptidespresent in their natural source.

The term “infection” refers to the introduction of nucleic acids into asuitable host cell by use of a virus or viral vector.

The term “transformation” means introducing DNA into a suitable hostcell so that the DNA is replicable, either as an extrachromosomalelement, or by chromosomal integration.

The term “transfection” refers to the taking up of an expression vectorby a suitable host cell, whether or not any coding sequences are in factexpressed.

The term “intermediate fragment” means a nucleic acid between about 5and about 1000 bases in length. In various embodiments, such nucleicacids are between about 10 and about 40 bp in length, or at least about100, 200, 300, 400, 20 500, 600, 700, 800 or 900 bp in length.

The term “secreted” protein refers to a protein that is transportedacross or through a membrane, including transport as a result of signalsequences in its amino acid sequence when it is expressed in a suitablehost cell. “Secreted” proteins include without limitation proteinssecreted wholly (e.g., soluble proteins) or partially (e.g., receptors,including seven-transmembrane receptors) from the cell in which they areexpressed. “Secreted” proteins also include without limitation proteinswhich are transported across the membrane of the endoplasmic reticulum.Each of the above terms is meant to encompasses all that is describedfor each, unless the context dictates otherwise.

Polynucleotides and Nucleic Acids of the Invention

Nucleotide and amino acid sequences of the invention are reported below.Fragments of the proteins of the present invention which are capable ofexhibiting biological activity are also encompassed by the presentinvention. Fragments of the protein may be in linear form or they may becyclized using known methods, for example, as described in H. U.saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference. Such fragments may be fusedto carrier molecules such as immunoglobulins for many purposes,including increasing the valency of protein binding sites. For example,fragments of the protein may be fused through “linker” sequences to theFc portion of an immunoglobulin. For a bivalent form of the protein,such a fusion could be to the Fc portion of an IgG molecule. Otherimmunoglobulin isotypes may also be used to generate such fusions. Forexample, a protein—IgM fusion would generate a decavalent form of theprotein of the invention.

The present invention also provides both full-length and mature forms ofthe disclosed proteins. The full-length forms of the polypeptides of theinvention are identified in the figures and the sequence listing bytranslation of the nucleotide sequence of each nucleic acid molecule.Mature forms of the polypeptides of the invention can routinely beobtained by expression of the disclosed nucleotides encoding thefull-length polypeptides in a suitable mammalian cell or other hostcell. The sequence of the mature forms of the polypeptides can alsoroutinely be determined from the amino acid sequence of the full-lengthpolypeptides.

The present invention also provides genes corresponding to cDNAsequences disclosed herein. The corresponding genes can be isolated inaccordance with known methods using the sequence information disclosedherein. Such methods include the preparation of probes or primers fromthe disclosed sequence information for identification and/oramplification of genes in appropriate genomic libraries or other sourcesof genomic materials.

Where the protein of the present invention is membrane-bound (e.g., is areceptor), the present invention also provides for soluble forms of suchprotein. In such forms part or all of the intracellular andtransmembrane domains of the protein are deleted such that the proteinis fully secreted from the cell in which it is expressed. Theintracellular and transmembrane domains of proteins of the invention canbe identified in accordance with known techniques for determination ofsuch domains from sequence information.

Species homologs of the disclosed polynucleotides and proteins are alsoprovided by the present invention. Species homologs may be isolated andidentified by making suitable probes or primers from the sequencesprovided herein and screening a suitable nucleic acid source from thedesired species. Species homologs can include, but are not limited tohuman, murine, rat or Drosophila species homologs.

The invention also encompasses allelic variants of the disclosedpolynucleotides or proteins; that is, naturally-occurring alternativeforms of the isolated polynucleotide which also encode proteins whichare identical, homologous or related to that encoded by thepolynucleotides. Sequences and allelic variant sequences of theinvention can include, but are not limited to human, murine, rat andDrosophila sequences.

The compositions of the present invention include isolatedpolynucleotides, including recombinant DNA molecules, cloned genes ordegenerate variants thereof, especially naturally occurring variantssuch as allelic variants, novel isolated polypeptides, and antibodiesthat specifically recognize one or more epitopes present on suchpolypeptides.

5.2. Nucleic Acids of the Invention

The isolated polynucleotides of the invention include, but are notlimited to, a polynucleotide encoding a polypeptide comprising the aminoacid sequence of SEQ ID NO:2 (also referred to herein as “CD39L2”); or apolynucleotide encoding a polypeptide comprising amino acid residues72-93, 147-162, 191-211 OR 217-238 of SEQ ID NO:2.

In selected embodiments, such isolated polynucleotides of the inventionrepresents a polynucleotide comprising the nucleotide sequence of SEQ IDNO:1; or a polynucleotide comprising nucleotides 232-1599, 445-510,670-717, 802-864 or 880-945 of the nucleotide sequence of SEQ ID NO:1.

The polynucleotides of the present invention also include, but are notlimited to, a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:1 under highly stringent hybridizationconditions; a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:1 under moderately stringenthybridization conditions; a polynucleotide which is an allelic variantof any polynucleotide recited above; a polynucleotide which encodes aspecies homologue of any of the proteins recited above; or apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptide of SEQ ID NO:2. Such polynucleotideshybridize under the above conditions to the complement of SEQ ID NO:1 orto a fragment of SEQ ID NO:1, wherein the fragment is greater than atleast about 10 bp, and, in alternate embodiments, is about 20 to about50 bp, or is greater than about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp,600 bp, 700 bp, or 800 bp.

The isolated polynucleotides of the invention still further include, butare not limited to, a polynucleotide encoding a polypeptide comprisingthe amino acid sequence of SEQ ID NO:4 (also referred to herein as“CD39L3”); or a polynucleotide encoding a polypeptide comprising aminoacid residues 55-76, 132-150, 179-199 or 213-234 of SEQ ID NO:4.

In selected embodiments, such isolated polynucleotides of the inventionrepresents a polynucleotide comprising the nucleotide sequence of SEQ IDNO:3; or a polynucleotide comprising nucleotides 83-1669, 245-310,476-532, 611-679 or 719-784 of the nucleotide sequence of SEQ ID NO:3.

The polynucleotides of the present invention also include, but are notlimited to, a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:3 under highly stringent hybridizationconditions; a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:3 under moderately stringenthybridization conditions; a polynucleotide which is an allelic variantof any polynucleotide recited above; a polynucleotide which encodes aspecies homologue of any of the proteins recited above; or apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptide of SEQ ID NO:4. Such polynucleotideshybridize under the above conditions to the complement of SEQ ID NO:3 orto a fragment of SEQ ID NO:3, wherein the fragment is greater than atleast about 10 bp, and, in alternate embodiments, is about 20 to about50 bp, or is greater than about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp,600 bp, 700 bp, or 800 bp.

The isolated polynucleotides of the invention still further include, butare not limited to, a polynucleotide encoding a polypeptide comprisingthe amino acid sequence of SEQ ID NO:6 (also referred to herein as“CD39L4”); or a polynucleotide encoding a polypeptide comprising aminoacid residues 47-68, 123-138, 167-187 or 193-214 of SEQ ID NO:6; or apolynucleotide encoding a polypeptide comprising the amino acid sequenceof SEQ ID NO:9 (also referred to herein as dCD39L4”); or apolynucleotide encoding amino acid residues 77-98, 153-167, 197-217 or223-242 of SEQ ID NO:9.

In one embodiment, such isolated polynucleotides of the inventionrepresents a polynucleotide comprising the nucleotide sequence of SEQ IDNO:5; or a polynucleotide comprising nucleotides 247-1530, 385-450,613-660, 745-807 or 823-888 of the nucleotide sequence of SEQ ID NO:5.

The polynucleotides of the present invention also include, but are notlimited to, a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:5 under highly stringent hybridizationconditions; a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:5 under moderately stringenthybridization conditions; a polynucleotide which is an allelic variantof any polynucleotide recited above; a polynucleotide which encodes aspecies homologue of any of the proteins recited above; or apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptide of SEQ IP NO:6. Such polynucleotideshybridize under the above conditions to the complement of SEQ ID NO:5 orto a fragment of SEQ ID NO:5, wherein the fragment is greater than atleast about 10 bp, and, In alternate embodiments, is about 20 to about50 bp, or is greater than about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp,600 bp, 700 bp or 800 bp.

The isolated polynucleotides of the invention further include, but arenot limited to, a polynucleotide encoding a polypeptide comprising theamino acid sequence of SEQ ID NO:8 (also referred to herein as “mCD39L4”or “mNTPase”); or a polynucleotide encoding amino acid residues 46-67,122-140, 166-187 or 194-213 of SEQ ID NO:8.

In selected embodiments, such isolated polynucleotides of the inventionrepresents a polynucleotide comprising the nucleotide sequence of SEQ IDNO:7; or a polynucleotide comprising nucleotides 205-1599, 340-395,568-624, 700-765 or 784-843 of the nucleotide sequence of SEQ ID NO:7.

The polynucleotides of the present invention also include, but are notlimited to, a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:7 under highly stringent hybridizationconditions; a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO:7 under moderately stringenthybridization conditions; a polynucleotide which is an allelic variantof any polynucleotide recited above; a polynucleotide which encodes aspecies homologue of any of the proteins recited above; or apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptide of SEQ ID NO:8. Such polynucleotideshybridize under the above conditions to the complement of SEQ ID NO:7 orto a fragment of SEQ ID NO:7, wherein the fragment is greater than atleast about 10 bp, and, in alternate embodiments, is about 20 to about50 bp, or is greater than about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp,600 bp, 700 bp or 800 bp.

The polynucleotides of the invention additionally include the complementof any of the polynucleotides recited above.

The polynucleotides of the invention also provide polynucleotides thatare substantially equivalent to the polynucleotides recited above.Typically, such a substantially equivalent sequence varies from one ofthose listed herein by no more than about 20%, i.e., the number ofindividual nucleotide substitutions, additions, and/or deletions in asubstantially equivalent sequence, as compared to the correspondingreference sequence, divided by the total number of nucleotides in thesubstantially equivalent sequence is about 0.2 or less. Such a sequenceis said to have 80% sequence identity to the listed sequence. Such asubstantially equivalent sequence can be routinely identified byapplying the foregoing algorithm.

In one embodiment, a substantially equivalent polynucleotide sequence ofthe invention varies from a listed sequence by no more than 10%, i.e.,the number of individual nucleotide substitutions, additions, and/ordeletions in a substantially equivalent sequence, as compared to thecorresponding reference sequence, divided by the total number ofnucleotides in the substantially equivalent sequence is about 0.1 orless. Such a sequence is said to have 90% sequence identity to thelisted sequence. Such a substantially equivalent sequence can beroutinely identified by applying the foregoing algorithm.

In an alternate embodiment a substantially equivalent sequence of theinvention varies from a listed sequence by no more than by no more than5%, i.e., the number of individual nucleotide substitutions, additions,and/or deletions in a substantially equivalent sequence, as compared tothe corresponding reference sequence, divided by the total number ofnucleotides in the substantially equivalent sequence is about 0.05 orless. Such a sequence is said to have 95% sequence identity to thelisted sequence. Such a substantially equivalent sequence can beroutinely identified by applying the foregoing algorithm.

In yet another alternate embodiment, a substantially equivalent sequenceof the invention varies from a listed sequences by no more than 2%,i.e., the number of individual nucleotide substitutions, additions,and/or deletions in a substantially equivalent sequence, as compared tothe corresponding reference sequence, divided by the total number ofnucleotides in the substantially equivalent sequence is about 0.02 orless. Such a sequence is said to have 98% sequence identity to thelisted sequence. Such a substantially equivalent sequence can beroutinely identified.

A polynucleotide according to the invention can be joined to any of avariety of other nucleotide sequences by well-established recombinantDNA techniques (see Sambrook J et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotidesequences for joining to polypeptides include an assortment of vectors,e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and thelike, that are well known in the art. Accordingly, the invention alsoprovides a vector including a polynucleotide of the invention and a hostcell containing the polynucleotide. In general, the vector contains anorigin of replication functional in at least one organism, convenientrestriction endonuclease sites, and a selectable marker for the hostcell. Vectors according to the invention include expression vectors,replication vectors, probe generation vectors, and sequencing vectors. Ahost cell according to the invention can be a prokaryotic or eukaryoticcell and can be a unicellular organism or part of a multicellularorganism.

The sequences falling within the scope of the present invention are notlimited to the specific sequences herein described, but also includeallelic variations thereof. Allelic variations can be routinelydetermined by comparing the sequence provided in SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5 or SEQ ID NO:7, a representative intermediate fragmentthereof, or a nucleotide sequence at least 99.9% identical to SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 with a sequence fromanother isolate of the same species. Furthermore, to accommodate codonvariability, the invention includes nucleic acid molecules coding forthe same amino acid sequences as do the specific ORFs disclosed herein.In other words, in the coding region of an ORF, substitution of onecodon for another which encodes the same amino acid is expresslycontemplated.

It is to be understood that nucleic acid molecules consisting of thefollowing nucleotide sequences are not considered part of the presentinvention: the nucleotide sequence or, where appropriate, the nucleotidesequence that encodes the depicted amino acid sequence, of Genbank™accession number S73813, gi11754710, U91510, U91511, AA116990, AA120757,H08436, AA378537, AA336644, AA338117, AA337885, N72742, AA256016,AA611283, AA647051, AA638277, AA271520, W46136, AA391695, AA390461,AA201196, AA246996, AA567512 or AC002032.

The present invention further provides recombinant constructs comprisinga nucleic acid having the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, or SEQ ID NO:7 or an intermediate fragment thereof, or another ofthe nucleic acid molecules of the invention. The recombinant constructsof the present invention comprise a vector, such as a plasmid or viralvector, into which a nucleic acid having the sequence of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7, or an intermediate fragmentthereof, or another of the nucleic acid molecules of the invention, isinserted, in a forward or reverse orientation. In the case of a vectorcomprising one of the ORFs of the present invention, the vector mayfurther comprise regulatory sequences, including for example, apromoter, operably linked to the ORF. For vectors comprising the EMFsand UMFs of the present invention, the vector may further comprise amarker sequence or heterologous ORF operably linked to the EMF or UMF.Large numbers of suitable vectors and promoters are known to those ofskill in the art and are commercially available for generating therecombinant constructs of the present invention. The following vectorsare provided by way of example. Bacterial: pBs, phagescript, PsiX174,pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene);pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic:pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL(Pharmacia).

The isolated polynucleotides of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490(1991), in order to produce the protein recombinantly. Many suitableexpression control sequences are known in the art. General methods ofexpressing recombinant proteins are also known and are exemplified in R.Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein“operably linked” means that the isolated polynucleotide of theinvention and an expression control sequence are situated within avector or cell in such a way that the protein is expressed by a hostcell which has been transformed (transfected) with the ligatedpolynucleotide/expression control sequence.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), andtrc. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art. Generally,recombinant expression vectors will include origins of replication andselectable markers permitting transformation of the host cell, e.g., theampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and apromoter derived from a highly-expressed gene to direct transcription ofa downstream structural sequence. Such promoters can be derived fromoperons encoding glycolytic enzymes such as 3-phosphoglycerate kinase(PGK), a-factor, acid phosphatase, or heat shock proteins, among others.The heterologous structural sequence is assembled in appropriate phasewith translation initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated proteininto the periplasmic space or extracellular medium. Optionally, theheterologous sequence can encode a fusion protein including anN-terminal identification peptide imparting desired characteristics,e.g., stabilization or simplified purification of expressed recombinantproduct. Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia PineChemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed. Followingtransformation of a suitable host strain and growth of the host strainto an appropriate cell density, the selected promoter is induced orderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification.

The nucleic acid sequences of the invention are further directed tosequences which encode variants of the described nucleic acids. Theseamino acid sequence variants may be prepared by methods known in the artby introducing appropriate nucleotide changes into a native or variantpolynucleotide. There are two variables in the construction of aminoacid sequence variants: the location of the mutation and the nature ofthe mutation. The amino acid sequence variants of the nucleic acids arepreferably constructed by mutating the polynucleotide to give an aminoacid sequence that does not occur in nature. These amino acidalterations can be made at sites that differ in the nucleic acids fromdifferent species (variable positions) or in highly conserved regions(constant regions). Sites at such locations will typically be modifiedin series, e.g., by substituting first with conservative choices (e.g.,hydrophobic amino acid to a different hydrophobic amino acid) and thenwith more distant choices (e.g., hydrophobic amino acid to a chargedamino acid), and then deletions or insertions may be made at the targetsite. Amino acid sequence deletions generally range from about 1 to 30residues, preferably about 1 to 10 residues, and are typicallycontiguous. Amino acid insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one to one hundred ormore residues, as well as intrasequence insertions of single or multipleamino acid residues. Intrasequence insertions may range generally fromabout 1 to 10 amino residues, preferably from 1 to 5 residues. Examplesof terminal insertions include the heterologous signal sequencesnecessary for secretion or for intracellular targeting in different hostcells.

In a preferred method, polynucleotides encoding the novel nucleic acidsare changed via site-directed mutagenesis. This method usesoligonucleotide sequences that encode the polynucleotide sequence of thedesired amino acid variant, as well as a sufficient adjacent nucleotideon both sides of the changed amino acid to form a stable duplex oneither side of the site of being changed. In general, the techniques ofsite-directed mutagenesis are well known to those of skill in the artand this technique is exemplified by publications such as, Edelman etal., DNA 2:183 (1983). A versatile and efficient method for producingsite-specific changes in a polynucleotide sequence was published byZoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may alsobe used to create amino acid sequence variants of the novel nucleicacids. When small amounts of template DNA are used as starting material,primer(s) that differs slightly in sequence from the correspondingregion in the template DNA can generate the desired amino acid variant.PCR amplification results in a population of product DNA fragments thatdiffer from the polynucleotide template encoding the polypeptide at theposition specified by the primer. The product DNA fragments replace thecorresponding region in the plasmid and this gives the desired aminoacid variant.

A further technique for generating amino acid variants is the cassettemutagenesis technique described in Wells et al., Gene 3:315 (1985); andother mutagenesis techniques well known in the art, such as, forexample, the techniques in Sambrook et al., supra, and Current Protocolsin Molecular Biology, Ausubel et al. Due to the inherent degeneracy ofthe genetic code, other DNA sequences which encode substantially thesame or a functionally equivalent amino acid sequence may be used in thepractice of the invention for the cloning and expression of these novelnucleic acids. Such DNA sequences include those which are capable ofhybridizing to the appropriate novel nucleic acid sequence understringent conditions.

Finally, it is to be understood that the nucleic acid molecules of theinvention further include any nucleic acid molecule that encodes thepolypeptides of the invention, as described in Section 5.4, below.

5.3. Hosts

The present invention further provides host cells genetically engineeredto contain the polynucleotides of the invention. For example, such hostcells may contain nucleic acids of the invention introduced into thehost cell using known transformation, transfection or infection methods.The present invention still further provides host cells geneticallyengineered to express the polynucleotides of the invention, wherein suchpolynucleotides are in operative association with a regulatory sequenceheterologous to the host cell which drives expression of thepolynucleotides in the cell.

The host cell can be a higher eukaryotic host cell, such as a mammaliancell, a lower eukaryotic host cell, such as a yeast cell, or the hostcell can be a prokaryotic cell, such as a bacterial cell. Introductionof the recombinant construct into the host cell can be effected bycalcium phosphate transfection, DEAE, dextran mediated transfection, orelectroporation (Davis, L. et al., Basic Methods in Molecular Biology(1986)). The host cells containing one of polynucleotides of theinvention, can be used in conventional manners to produce the geneproduct encoded by the isolated fragment (in the case of an ORF) or canbe used to produce a heterologous protein under the control of the EDF.

Any host/vector system can be used to express one or more of the ORFs ofthe present invention. These include, but are not limited to, eukaryotichosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9 cells, as wellas prokaryotic host such as E. coli and B. subtilis. The most preferredcells are those which do not normally express the particular polypeptideor protein or which expresses the polypeptide or protein at low naturallevel. Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al., inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989), the disclosure of which is hereby incorporated byreference.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell tines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and also any necessary ribosome binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking nontranscribed sequences. DNAsequences derived from the SV40 viral genome, for example, SV40 origin,early promoter, enhancer, splice, and polyadenylation sites may be usedto provide the required nontranscribed genetic elements. Recombinantpolypeptides and proteins produced in bacterial culture are usuallyisolated by initial extraction from cell pellets, followed by one ormore salting-out, aqueous ion exchange or size exclusion chromatographysteps. Protein refolding steps can be used, as necessary, in completingconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed for final purification steps.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

A number of types of cells may act as suitable host cells for expressionof the protein. Mammalian host cells include, for example, monkey COScells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, humanepidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, othertransformed primate cell lines, normal diploid cells, cell strainsderived from in vitro culture of primary tissue, primary explants, HeLacells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.

Alternatively, it may be possible to produce the protein in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Potentiallysuitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous proteins. Potentially suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous proteins. If the protein is made in yeast or bacteria, itmay be necessary to modify the protein produced therein, for example byphosphorylation or glycosylation of the appropriate sites, in order toobtain the functional protein. Such covalent attachments may beaccomplished using known chemical or enzymatic methods.

In another embodiment of the present invention, cells and tissues may beengineered to express an endogenous gene comprising the polynucleotidesof the invention under the, control of inducible regulatory elements, inwhich case the regulatory sequences of the endogenous gene may bereplaced by homologous recombination. As described herein, genetargeting can be used to replace a gene's existing regulatory regionwith a regulatory sequence isolated from a different gene or a novelregulatory sequence synthesized by genetic engineering methods. Suchregulatory sequences may be comprised of promoters, enhancers,scaffold-attachment regions, negative regulatory elements,transcriptional initiation sites, regulatory protein binding sites orcombinations of said sequences. Alternatively, sequences which affectthe structure or stability of the RNA or protein produced may bereplaced, removed, added, or otherwise modified by targeting, includingpolyadenylation signals. mRNA stability elements, splice sites, leadersequences for enhancing or modifying transport or secretion propertiesof the protein, or other sequences which alter or improve the functionor stability of protein or RNA molecules.

The targeting event may be a simple insertion of the regulatorysequence, placing the gene under the control of the new regulatorysequence, e.g., inserting a new promoter or enhancer or both upstream ofa gene. Alternatively, the targeting event may be a simple deletion of aregulatory element, such as the deletion of a tissue-specific negativeregulatory element. Alternatively, the targeting event may replace anexisting element; for example, a tissue-specific enhancer can bereplaced by an enhancer that has broader or different cell-typespecificity than the naturally occurring elements. Here, the naturallyoccurring sequences are deleted and new sequences are added. In allcases, the identification of the targeting event may be facilitated bythe use of one or more selectable marker genes that are contiguous withthe targeting DNA, allowing for the selection of cells in which theexogenous DNA has integrated into the host cell genome. Theidentification of the targeting event may also be facilitated by the useof one or more marker genes exhibiting the property of negativeselection, such that the negatively selectable marker is linked to theexogenous DNA, but configured such that the negatively selectable markerflanks the targeting sequence, and such that a correct homologousrecombination event with sequences in the host cell genome does notresult in the stable integration of the negatively selectable marker.Markers useful for this purpose include the Herpes Simplex Virusthymidine kinase (TK) gene or the bacterial xanthine-guaninephosphoribosyl-transferase (gpt) gene.

The gene targeting or gene activation techniques which can be used inaccordance with this aspect of the invention are more particularlydescribed in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461to Sherwin et al.; International Application No. PCT/US92/09627(WO93/09222) by Selden et al.; and International Application No.PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which isincorporated by reference herein in its entirety.

5.4. Polyeptides of the Invention

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising the amino acid sequence of SEQ ID NO:2; ora polypeptide comprising amino acid residues 72-93, 147-162, 191-211 OR217-238 of SEQ ID NO:2.

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising the amino acid sequence of SEQ ID NO:4; ora polypeptide comprising amino acid residues 55-76, 132-150, 179-199 or213-234 of SEQ ID NO:4.

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising the amino acid sequence of SEQ ID NO:6; ora polypeptide comprising amino acid residues 47-68, 123-138, 167-187 or193-214 of SEQ ID NO:6.

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising the amino acid sequence of SEQ ID NO:8; ora polypeptide comprising amino acid residues 46-67, 122-140, 166-187 or194-213 of SEQ ID NO:8.

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising the amino acid sequence of SEQ ID NO:9; ora polypeptide comprising amino acid residues 77-98, 153-167, 197-217 or223-242 of SEQ ID NO:9.

The isolated polypeptides of the invention further include polypeptidesthat are substantially equivalent to the polypeptides of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:9 or to specificdomains thereof. Typically, sucha substantially equivalent sequencevaries from one of those listed herein by no more than about 20%, i.e.,the number of individual amino acid residue substitutions, additions,and/or deletions in a substantially equivalent sequence, as compared tothe corresponding reference sequence, divided by the total number ofamino acid residues in the substantially equivalent sequence is about0.2 or less. Such a sequence is said to have 80% sequence identity tothe listed sequence. Such a substantially equivalent sequence can beroutinely identified by applying the foregoing algorithm.

In one embodiment, a substantially equivalent polypeptide sequence ofthe invention varies from a listed sequence by no more than 10%, i.e.,the number of individual amino acid substitutions, additions, and/ordeletions in a substantially equivalent sequence, as compared to thecorresponding reference sequence, divided by the total number of aminoacid residues in the substantially equivalent sequence is about 0.1 orless. Such a sequence is said to have 90% sequence identity to thelisted sequence. Such a substantially equivalent sequence can beroutinely identified by applying the foregoing algorithm.

In an alternate embodiment a substantially equivalent sequence of theinvention varies from a listed sequence by no more than by no more than5%, i.e., the number of individual amino acid substitutions, additions,and/or deletions in a substantially equivalent sequence, as compared tothe corresponding reference sequence, divided by the total number ofamino acid residues in the substantially equivalent sequence is about0.05 or less. Such a sequence is said to have 95% sequence identity tothe listed sequence. Such a substantially equivalent sequence can beroutinely identified by applying the foregoing algorithm.

In yet another alternate embodiment, a substantially equivalent sequenceof the invention varies from a listed sequences by no more than 2%,i.e., the number of individual amino acid residue substitutions,additions, and/or deletions in a substantially equivalent sequence, ascompared to the corresponding reference sequence, divided by the totalnumber of amino acid residues in the substantially equivalent sequenceis about 0.02 or less. Such a sequence is said to have 98% sequenceidentity to the listed sequence. Such a substantially equivalentsequence can be routinely identified.

Preferred embodiments include those in which the protein produced bysuch process is a mature form of the protein.

Protein compositions of the present invention may further comprise anacceptable carrier, such as a hydrophilic, e.g., pharmaceuticallyacceptable, carrier.

The invention also relates to methods for producing a polypeptidecomprising growing a culture of the cells of the invention in a suitableculture medium, and purifying the protein from the culture. For example,the methods of the invention include a process for producing apolypeptide in which a host cell containing a suitable expression vectorthat includes a polynucleotide of the invention is cultured underconditions that allow expression of the encoded polypeptide. Thepolypeptide can be recovered from the culture, conveniently from theculture medium, and further purified.

The present invention further provides isolated polypeptides encoded bythe nucleic acid fragments of the present invention or by degeneratevariants of the nucleic acid fragments of the present invention. By“degenerate variant” is intended nucleotide fragments which differ froma nucleic acid fragment of the present invention (e.g., an ORF) bynucleotide sequence but, due to the degeneracy of the genetic code,encode an identical polypeptide sequence. Preferred nucleic acidfragments of the present invention are the ORFs that encode proteins.

A variety of methodologies known in the art can be utilized to obtainany one of the isolated polypeptides or proteins of the presentinvention. At the simplest level, the amino acid sequence can besynthesized using commercially available peptide synthesizers. This isparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the native polypeptide. In an alternative method, thepolypeptide or protein is purified from bacterial cells which naturallyproduce the polypeptide or protein. One skilled in the art can readilyfollow known methods for isolating polypeptides and proteins in order toobtain one of the isolated polypeptides or proteins of the presentinvention. These include, but are not limited to, immunochromatography,HPLC, size-exclusion chromatography, ion-exchange chromatography, andimmuno-affinity chromatography. See, e.g., scopes, Protein Purification:Principles and Practice, Springer-Verlag (1994); Sambrook, et al., inMolecular Cloning: A Laboratory Manual; Ausubel et al., CurrentProtocols in Molecular Biology.

The polypeptides and proteins of the present invention can alternativelybe purified from cells which have been altered to express the desiredpolypeptide or protein. As used herein, a cell is said to be altered toexpress a desired polypeptide or protein when the cell, through geneticmanipulation, is made to produce a polypeptide or protein which itnormally does not produce or which the cell normally produces at a lowerlevel. One skilled in the art can readily adapt procedures forintroducing and expressing either recombinant or synthetic sequencesinto eukaryotic or prokaryotic cells in order to generate a cell whichproduces one of the polypeptides or proteins of the present invention.The purified polypeptides can be used in in vitro binding assays whichare well known in the art to identify molecules which bind to thepolypeptides. These molecules include but are not limited to, for e.g.,small molecules, molecules from combinatorial libraries, antibodies orother proteins. The molecules identified in the binding assay are thentested for antagonist or agonist activity in in vivo tissue culture oranimal models that are well known in the art. In brief, the moleculesare titrated into a plurality of cell cultures or animals and thentested for either cell/animal death or prolonged survival of theanimal/cells.

In addition, the binding molecules may be complexed with toxins, e.g.,ricin or cholera, or with other compounds that are toxic to cells. Thetoxin-binding molecule complex is then targeted to the tumor or othercell by the specificity of the binding molecule for SEQ ID NO:2, SEQ IDNO:4 SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:9, or another of thepolypeptide of the invention.

The protein of the invention may also be expressed as a product oftransgenic animals, e.g., as a component of the milk of transgenic cows,goats, pigs, or sheep which are characterized by somatic or germ cellscontaining a nucleotide sequence encoding the protein.

The protein may also be produced by known conventional chemicalsynthesis. Methods for constructing the proteins of the presentinvention by synthetic means are known to those skilled in the art. Thesynthetically-constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with proteins may possess biological properties incommon therewith, including protein activity. Thus, they may be employedas biologically active or immunological substitutes for natural,purified proteins in screening of therapeutic compounds and inimmunological processes for the development of antibodies.

The proteins provided herein also include proteins characterized byamino acid sequences similar to those of purified proteins but intowhich modification are naturally provided or deliberately engineered.For example, modifications in the peptide or DNA sequences can be madeby those skilled in the art using known techniques. Modifications ofinterest in the protein sequences may include the alteration,substitution, replacement, insertion or deletion of a selected aminoacid residue in the coding sequence. For example, one or more of thecysteine residues may be deleted or replaced with another amino acid toalter the conformation of the molecule. Techniques for such alteration,substitution, replacement, insertion or deletion are well known to thoseskilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably,such alteration, substitution, replacement, insertion or deletionretains the desired activity of the protein.

Other fragments and derivatives of the sequences of proteins which wouldbe expected to retain protein activity in whole or in part and may thusbe useful for screening or other immunological methodologies may also beeasily made by those skilled in the art given the disclosures herein.Such modifications are believed to be encompassed by the presentinvention.

The protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif., U.S.A. (the MaxBat.RTH. kit), and such methods arewell known in the art, as described in Summers and Smith, TexasAgricultural Experiment Station Bulletin No. 1555 (1987), incorporatedherein by reference. As used herein, an insect cell capable ofexpressing a polynucleotide of the present invention is “transformed.”

The protein of the invention may be prepared by culturing transformedhost cells under culture conditions suitable to express the recombinantprotein. The resulting expressed protein may then be purified from suchculture (i.e., from culture medium or cell extracts) using knownpurification processes, such as gel filtration and ion exchangechromatography. The purification of the protein may also include anaffinity column containing agents which will bind to the protein; one ormore column steps over such affinity resins as concanavalin A-agarose,heparin-toyopearl.RTM. or Cibacrom blue 3GA Sepharose.RTM.; one or moresteps involving hydrophobic interaction chromatography using such resinsas phenyl ether, butyl ether, or propyl ether; or immunoaffinitychromatography.

Alternatively, the protein of the invention may also be expressed in aform which will facilitate purification. For example, it may beexpressed as a fusion protein, such as those of maltose binding protein(MBP), glutathione-s-transferase (GST) or thioredoxin (TRX). Kits forexpression and purification of such fusion proteins are commerciallyavailable from New England BioLab (Beverly, Mass.), Pharmacia(Piscataway, N.J.) and In Vitrogen, respectively. The protein can alsobe tagged with an epitope and subsequently purified by using a specificantibody directed to such epitope. One such epitope (“Flag”) iscommercially available from Kodak (New Haven, Conn.).

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the protein. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a substantially homogeneous isolated recombinant protein. Theprotein thus purified is substantially free of other mammalian proteinsand is defined in accordance with the present invention as an “isolatedprotein.”

It is to be understood that polypeptides consisting of the followingamino acid sequence are not considered part of the present invention:the amino acid sequence of, or, where appropriate, reported to beencoded by the nucleotide sequence of Genbank™ accession No: S73813,gi11754710, U91510, U91511, AA116990, AA120757, H08436, AA378537,AA336644, AA338117, AA337885, N72742, AA256016, AA611283, AA647051,AA638277, AA271520, W46136, AA391695, AA390461, AA201196, AA246996 orAA567512.

5.5. Uses and Biological Activity

The polynucleotides and proteins of the present invention are expectedto exhibit one or more of the uses or biological activities (includingthose associated with assays cited herein) identified below. Uses oractivities described for proteins of the present invention may beprovided by administration or use of such proteins or by administrationor use of polynucleotides encoding such proteins (such as, for example,in gene therapies or vectors suitable for introduction of DNA).

5.5.1. Research Uses and Utilities

The polynucleotides provided by the present invention can be used by theresearch community for various purposes. The polynucleotides can be usedto express recombinant protein for analysis, characterization ortherapeutic use; as markers for tissues in which the correspondingprotein is preferentially expressed (either constitutively or at aparticular stage of tissue differentiation or development or in diseasestates); as molecular weight markers on Southern gels; as chromosomemarkers or tags (when labeled) to identify chromosomes or to map relatedgene positions; to compare with endogenous DNA sequences in patients toidentify potential genetic disorders; as probes to hybridize and thusdiscover novel, related DNA sequences; as a source of information toderive PCR primers for genetic fingerprinting; as a probe to“subtract-out” known sequences in the process of discovering other novelpolynucleotides; for selecting and making oligomers for attachment to a“gene chip” or other support, including for examination of expressionpatterns; to raise anti-protein antibodies using DNA immunizationtechniques; and as an antigen to raise anti-DNA antibodies or elicitanother immune response. Where the polynucleotide encodes a proteinwhich binds or potentially binds to another protein (such as, forexample, in a receptor-ligand interaction), the polynucleotide can alsobe used in interaction trap assays (such as, for example, that describedin Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotidesencoding the other protein with which binding occurs or to identifyinhibitors of the binding interaction.

The proteins provided by the present invention can similarly be used inassay to determine biological activity, including in a panel of multipleproteins for high-throughput screening; to raise antibodies or to elicitanother immune response; as a reagent (including the labeled reagent) inassays designed to quantitatively determine levels of the protein (orits receptor) in biological fluids; as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state); and, of course, to isolate correlative receptors orligands. Where the protein binds or potentially binds to another protein(such as, for example, in a receptor-ligand interaction), the proteincan be used to identify the other protein with which binding occurs orto identify inhibitors of the binding interaction. Proteins involved inthese binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction.

Any or all of these research utilities are capable of being developedinto reagent grade or kit format for commercialization as researchproducts.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include withoutlimitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold springHarbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatiseds., 1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

5.5.2. Nutritional Uses

Polynucleotides and proteins of the present invention can also be usedas nutritional sources or supplements. Such uses include withoutlimitation use as a protein or amino acid supplement, use as a carbonsource, use as a nitrogen source and use as a source of carbohydrate. Insuch cases the protein or polynucleotide of the invention can be addedto the feed of a particular organism or can be administered as aseparate solid or liquid preparation, such as in the form of powder,pills, solutions, suspensions or capsules. In the case ofmicroorganisms, the protein or polynucleotide of the invention can beadded to the medium in or on which the microorganism is cultured.

5.5.3. Cytokine and Cell Proliferation/Differentiation Activity

A protein of the present invention may exhibit cytokine, cellproliferation (either inducing or inhibiting) or cell differentiation(either inducing or inhibiting) activity or may induce production ofother cytokines in certain cell populations. A polynucleotide of theinvention can encode a polypeptide exhibiting such attributes. Manyprotein factors discovered to date, including all known cytokines, haveexhibited activity in one or more factor-dependent cell proliferationassays, and hence the assays serve as a convenient confirmation ofcytokine activity. The activity of a protein of the present invention isevidenced by any one of a number of routine factor dependent cellproliferation assays for cell lines including, without limitation, 32D,DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123,T1165, HT2, CTLL2, TF-1, Mo7e and CMX.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for T-cell or thymocyte proliferation include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, InVitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolliet al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I.Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761,1994.

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek, A. M. andShevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coliganeds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; andMeasurement of mouse and human interleukin gamma., Schreiber, R. D. InCurrent Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Measurement of Human and Murine Interleukin 2 and Interleukin 4,Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols inImmunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wileyand Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211,1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse andhuman interleukin 6—Nordan, R. In Current Protocols in Immunology. J. E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto.1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986;Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark,S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e.a.Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti,J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology.J. E. e.a. Coligan ads. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.1991.

Assays for T-cell clone responses to antigens (which will Identify,among others, proteins that affect APC-T cell interactions as well asdirect T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction; Chapter 6, Cytokines and their cellular receptors; Chapter 7,Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad.Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.11:405-411. 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takaiet al., J. Immunol. 140:508-512, 1988.

5.5.4. Immune Stimulating or Suppressing Activity

A protein of the present invention may also exhibit immune stimulatingor immune suppressing activity, including without limitation theactivities for which assays are described herein. A polynucleotide ofthe invention can encode a polypeptide exhibiting such activities. Aprotein may be useful in the treatment of various immune deficienciesand disorders (including severe combined immunodeficiency (SCID)), e.g.,in regulating (up or down) growth and proliferation of T and/or Blymphocytes, as well as effecting the cytolytic activity of NK cells andother cell populations. These immune deficiencies may be genetic or becaused by vital (e.g., HIV) as well as bacterial or fungal infections,or may result from autoimmune disorders. More specifically, infectiousdiseases causes by viral, bacterial, fungal or other infection may betreatable using a protein of the present invention, including infectionsby HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp.,malaria spp. and various fungal infections such as candidiasis. Ofcourse, in this regard, a protein of the present invention may also beuseful where a boost to the immune system generally may be desirable,i.e., in the treatment of cancer.

Autoimmune disorders which may be treated using a protein of the presentinvention include, for example, connective tissue disease, multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis,autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunethyroiditis, insulin dependent diabetes mellitis, myasthenia gravis,graft-versus-host disease and autoimmune inflammatory eye disease. Sucha protein of the present invention may also to be useful in thetreatment of allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems. Otherconditions, in which immune suppression is desired (including, forexample, organ transplantation), may also be treatable using a proteinof the present invention.

Using the proteins of the invention it may also be possible to immuneresponses, in a number of ways. Down regulation may be in the form ofinhibiting or blocking an immune response already in progress or mayinvolve preventing the induction of an immune response. The functions ofactivated T cells may be inhibited by suppressing T cell responses or byinducing specific tolerance in T cells, or both. Immunosuppression of Tcell responses is generally an active, non-antigen-specific, processwhich requires continuous exposure of the T cells to the suppressiveagent. Tolerance, which involves inducing non-responsiveness or anergyin T cells, is distinguishable from immunosuppression in that it isgenerally antigen-specific and persists after exposure to the tolerizingagent has ceased. Operationally, tolerance can be demonstrated by thelack of a T cell response upon reexposure to specific antigen in theabsence of the tolerizing agent.

Down regulating or preventing one or more antigen functions (includingwithout limitation B lymphocyte antigen functions (such as, for example,B7)), e.g., preventing high level lymphokine synthesis by activated Tcells, will be useful in situations of tissue, skin and organtransplantation and in graft-versus-host disease (GVHD). For example,blockage of T cell function should result in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated through its recognition as foreign by Tcells, followed by an immune reaction that destroys the transplant. Theadministration of a molecule which inhibits or blocks interaction of aB7 lymphocyte antigen with its natural ligand(s) on immune cells (suchas a soluble, monomeric form of a peptide having B7-2 activity alone orin conjunction with a monomeric form of a peptide having an activity ofanother B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody),prior to transplantation can lead to the binding of the molecule to thenatural ligand(s) on the immune cells without transmitting thecorresponding costimulatory signal. Blocking B lymphocyte antigenfunction in this matter prevents cytokine synthesis by immune cells,such as T cells, and thus acts as an immunosuppressant. Moreover, thelack of costimulation may also be sufficient to anergize the T cells,thereby inducing tolerance in a subject. Induction of long-termtolerance by B lymphocyte antigen-blocking reagents may avoid thenecessity of repeated administration of these blocking reagents. Toachieve sufficient immunosuppression or tolerance in a subject, it mayalso be necessary to block the function of a combination of B lymphocyteantigens.

The efficacy of particular blocking reagents in preventing organtransplant rejection or GVHD can be assessed using animal models thatare predictive of efficacy in humans. Examples of appropriate systemswhich can be used include allogeneic cardiac grafts in rats andxenogeneic pancreatic islet cell grafts in mice, both of which have beenused to examine the immunosuppressive effects of CTLA4Ig fusion proteinsin vivo as described in Lenschow et al., Science 257:789-792 (1992) andTurka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). Inaddition, murine models of GVHD (see Paul ed., Fundamental Immunology,Raven Press, New York, 1989, pp. 846-847) can be used to determine theeffect of blocking B lymphocyte antigen function in vivo on thedevelopment of that disease.

Blocking antigen function may also be therapeutically useful fortreating autoimmune diseases. Many autoimmune disorders are the resultof inappropriate activation of T cells that are reactive against selftissue and which promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive T cells may reduce or eliminate disease symptoms.Administration of reagents which block costimulation of T cells bydisrupting receptor:ligand interactions of B lymphocyte antigens can beused to inhibit T cell activation and prevent production ofautoantibodies or T cell-derived cytokines which may be involved in thedisease process. Additionally, blocking reagents may induceantigen-specific tolerance of autoreactive T cells which could lead tolong-term relief from the disease. The efficacy of blocking reagents inpreventing or alleviating autoimmune disorders can be determined using anumber of well-characterized animal models of human autoimmune diseases.Examples include murine experimental autoimmune encephalitis, systemiclupus erythmatosis in MRL/1pr/1pr mice or NZB hybrid mice, murineautoimmune collagen arthritis, diabetes mellitus in NOD mice and BBrats, and murine experimental myasthenia gravis (see Paul ed.,Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Upregulation of an antigen function (preferably a B lymphocyte antigenfunction), as a means of up regulating immune responses, may also beuseful in therapy. Upregulation of immune responses may be in the formof enhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response through stimulatingB lymphocyte antigen function may be useful in cases of viral infection.In addition, systemic viral diseases such as influenza, the common cold,and encephalitis might be alleviated by the administration ofstimulatory forms of B lymphocyte antigens systemically.

Alternatively, anti-vital immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide of the present invention or together with a stimulatory form ofa soluble peptide of the present invention and reintroducing the invitro activated T cells into the patient. Another method of enhancinganti-viral immune responses would be to isolate infected cells from apatient, transfect them with a nucleic acid encoding a protein of thepresent invention as described herein such that the cells express all ora portion of the protein on their surface, and reintroduce thetransfected cells into the patient. The infected cells would now becapable of delivering a costimulatory signal to, and thereby activate, Tcells in vivo.

The presence of the peptide of the present invention having the activityof a B lymphocyte antigen(s) on the surface of the tumor cell providesthe necessary costimulation signal to T cells to induce a T cellmediated immune response against the transfected tumor cells. Inaddition, tumor cells which lack MHC class I or MHC class II molecules,or which fail to reexpress sufficient mounts of MHC class I or MHC classII molecules, can be transfected with nucleic acid encoding all or aportion of (e.g., a cytoplasmic-domain truncated portion) of an MHCclass I a .alpha. chain protein and beta.sub.2 microglobulin protein oran MHC class II alpha. chain protein and an MHC class II .beta. chainprotein to thereby express MHC class I or MHC class II proteins on thecell surface. Expression of the appropriate class I or class II MHC inconjunction with a peptide having the activity of a B lymphocyte antigen(e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune responseagainst the transfected tumor cell. Optionally, a gene encoding anantisense construct which blocks expression of an MHC class IIassociated protein, such as the invariant chain, can also becotransfected with a DNA encoding a peptide having the activity of a Blymphocyte antigen to promote presentation of tumor associated antigensand induce tumor specific immunity. Thus, the induction of a T cellmediated immune response in a human subject may be sufficient toovercome tumor-specific tolerance in the subject.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include,without limitation, those described in: Current Protocols in Immunology,Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985;Takai et al., I. Immunol. 137:3494-3500, 1986; Takai et-al., J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982;Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol.137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai etal., J. Immunol. 140:508-512, 1988; Bertagnolli et al., CellularImmunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092,1994.

Assays for T-cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, proteins that modulateT-cell dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski, J.Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitroantibody production, Mond, J. J. and Brunswick, M. In Current Protocolsin Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, JohnWiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, amongothers, proteins that generate predominantly Th1 and CTL responses)include, without limitation, those described in: Current Protocols inImmunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.Shevach, W. Strober, Pub. Greene Publishing Associates andWiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai etal., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

Dendritic cell-dependent assays (which will identify, among others,proteins expressed by dendritic cells that activate naive T-cells)include, without limitation, those described in: Guery et al., J.Immunol. 134:536-544, 1995; Inaba et al., Journal of ExperimentalMedicine 173:549-559, 1991; Macatonia et al., Journal of Immunology154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993;Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal ofExperimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal ofClinical Investigation 94:797-807, 1994; and Inaba et al., Journal ofExperimental Medicine 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, amongothers, proteins that prevent apoptosis after superantigen induction andproteins that regulate lymphocyte homeostasis) include, withoutlimitation, those described in: Darzynkiewicz et al., Cytometry13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorozyca etal., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243,1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai etal., Cytometry 14:891-897, 1993; Gorczyca et al., International Journalof Oncology 1:639-648, 1992.

Assays for proteins that influence early steps of T-cell commitment anddevelopment include, without limitation, those described in: Antica etal., Blood 84:111-117, 1994; Fine et al., Cellular Immunology155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al.,Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.

5.5.5. Hematopoiesis Regulating Activity

A protein of the present invention may be useful in regulation ofhematopoiesis and, consequently, in the treatment of myeloid or lymphoidcell deficiencies. Even marginal biological activity in support ofcolony forming cells or of factor-dependent cell lines indicatesinvolvement in regulating hematopoiesis, e.g. in supporting the growthand proliferation of erythroid progenitor cells alone or in combinationwith other cytokines, thereby indicating utility, for example, intreating various anemias or for use in conjunction withirradiation/chemotherapy to stimulate the production of erythroidprecursors and/or erythroid cells; in supporting the growth andproliferation of myeloid cells such as granulocytes andmonocytes/macrophages (i.e., traditional CSF activity) useful, forexample, in conjunction with chemotherapy to prevent or treat consequentmyelo-suppression; in supporting the growth and proliferation ofmegakaryocytes and consequently of platelets thereby allowing preventionor treatment of various platelet disorders such as thrombocytopenia, andgenerally for use in place of or complimentary to platelet transfusions;and/or in supporting the growth and proliferation of bematopoietic stemcells which are capable of maturing to any and all of theabove-mentioned hematopoietic cells and therefore find therapeuticutility in various stem cell disorders (such as those usually treatedwith transplantation, including, without limitation, aplastic anemia andparoxysmal nocturnal hemoglobinuria), as well as in repopulating thestem cell compartment post irradiation/chemotherapy, either in-vivo orex-vivo (i.e., in conjunction with bone marrow transplantation or withperipheral progenitor cell transplantation (homologous or heterologous))as normal cells or genetically manipulated for gene therapy.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for proliferation and differentiation of varioushematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify,among others, proteins that influence embryonic differentiationhematopoiesis) include, without limitation, those described in:Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al.,Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,Blood 81:2903-2915, 1993.

Assays for stem cell survival and differentiation (which will identify,among others, proteins that regulate lympho-hematopoiesis) include,without limitation, those described in: Methylcellulose colony formingassays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;Primitive hematopoietic colony forming cells with high proliferativepotential, McNiece, I. K. and Briddell, R. A. In Culture ofHematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., ExperimentalHematology 22:353-359, 1994; Cobblestone area forming cell assay,Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, etal. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long termbone marrow cultures in the presence of stromal cells, Spooncer, E.,Dexter, M. and Allen, T. In Culture of Hematopoiatic Cells. R. I.Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y.1994; Long term culture initiating cell assay, Sutherland, H. J. InCulture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

5.5.6. Tissue Growth Activity

A protein of the present invention also may have utility in compositionsused for bone, cartilage, tendon, ligament and/or nerve tissue growth orregeneration, as well as for wound healing and tissue repair andreplacement, and in the treatment of burns, incisions and ulcers.

A protein of the present invention, which induces cartilage and/or bonegrowth in circumstances where bone is not normally formed, hasapplication in the healing of bone fractures and cartilage damage ordefects in humans and other animals. Such a preparation employing aprotein of the invention may have prophylactic use in closed as well asopen fracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery.

A protein of this invention may also be used in the treatment ofperiodontal disease, and in other tooth repair processes. Such agentsmay provide an environment to attract bone-forming cells, stimulategrowth of bone-forming cells or induce differentiation of progenitors ofbone-forming cells. A protein of the invention may also be useful in thetreatment of osteoporosis or osteoarthritis, such as through stimulationof bone and/or cartilage repair or by blocking inflammation or processesof tissue destruction (collagenase activity, osteoclast activity, etc.)mediated by inflammatory processes.

Another category of tissue regeneration activity that may beattributable to the protein of the present invention is tendon/ligamentformation. A protein of the present invention, which inducestendon/ligament-like tissue or other tissue formation in circumstanceswhere such tissue is not normally formed, has application in the healingof tendon or ligament tears, deformities and other tendon or ligamentdefects in humans and other animals. Such a preparation employing atendon/ligament-like tissue inducing protein may have prophylactic usein preventing damage to tendon or ligament tissue, as well as use in theimproved fixation of tendon or ligament to bone or other tissues, and inrepairing defects to tendon or ligament tissue. De novotendon/ligament-like tissue formation induced by a composition of thepresent invention contributes to the repair of congenital, traumainduced, or other tendon or ligament defects of other origin, and isalso useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the present invention mayprovide environment to attract tendon- or ligament-forming cells,stimulate growth of tendon- or ligament-forming cells, inducedifferentiation of progenitors of tendon- or ligament-forming cells, orinduce growth of tendon/ligament cells or progenitors ex vivo for returnin vivo to effect tissue repair. The compositions of the invention mayalso be useful in the treatment of tendinitis, carpal tunnel syndromeand other tendon or ligament defects. The compositions may also includean appropriate matrix and/or sequestering agent as a carrier as is wellknown in the art.

The protein of the present invention may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue, i.e. for the treatment of central and peripheral nervous systemdiseases and neuropathies, as well as mechanical and traumaticdisorders, which involve degeneration, death or trauma to neural cellsor nerve tissue. More specifically, a protein may be used in thetreatment of diseases of the peripheral nervous system, such asperipheral nerve injuries, peripheral neuropathy and localizedneuropathies, and central nervous system diseases, such as Alzheimer's,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome. Further conditions which may betreated in accordance with the present invention include mechanical andtraumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a protein of the invention.

Proteins of the invention may also be useful to promote better or fasterclosure of non-healing wounds, including without limitation pressureulcers, ulcers associated with vascular insufficiency, surgical andtraumatic wounds, and the like.

It is expected that a protein of the present invention may also exhibitactivity for generation or regeneration of other tissues, such as organs(including, for example, pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac) and vascular(including vascular endothelium) tissue, or for promoting the growth ofcells comprising such tissues. Part of the desired effects may be byinhibition or modulation of fibrotic scarring to allow normal tissue toregenerate. A protein of the invention may also exhibit angiogenicactivity.

A protein of the present invention may also be useful for gut protectionor regeneration and treatment of lung or liver fibrosis, reperfusioninjury in various tissues, and conditions resulting from systemiccytokine damage.

A protein of the present invention may also be useful for promoting orinhibiting differentiation of tissues described above from precursortissues or cells; or for inhibiting the growth of tissues describedabove.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for tissue generation activity include, without limitation, thosedescribed in: International Patent Publication No. WO95/16035 (bone,cartilage, tendon); International Patent Publication No. WO95/05846(nerve, neuronal); International Patent Publication No. WO91/07491(skin, endothelium).

Assays for wound healing activity include, without limitation, thosedescribed in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H.I. and Rovee, D. T., eds.), Year Book Medical Publishers, Inc., Chicago,as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84(1978).

5.5.7. Activin/Inhibin Activity

A protein of the present invention may also exhibit activin- orinhibin-related activities. A polynucleotide of the invention may encodea polypeptide exhibiting such characteristics. Inhibins arecharacterized by their ability to inhibit the release of folliclestimulating hormone (FSH), while activins and are characterized by theirability to stimulate the release of follicle stimulating hormone (FSH).Thus, a protein of the present invention, alone or in heterodimers witha member of the inhibin α-family, may be useful as a contraceptive basedon the ability of inhibins to decrease fertility in female mammals anddecrease spermatogenesis in male mammals. Administration of sufficientamounts of other inhibins can induce infertility in these mammals.Alternatively, the protein of the invention, as a homodimer or as aheterodimer with other protein subunits of the inhibin-β group, may beuseful as a fertility inducing therapeutic, based upon the ability ofactivin molecules in stimulating FSH release from cells of the anteriorpituitary. See, for example, U.S. Pat. No. 4,798,885. A protein of theinvention may also be useful for advancement of the onset of fertilityin sexually immature mammals, so as to increase the lifetimereproductive performance of domestic animals such as cows, sheep andpigs.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for activin/inhibin activity include, without limitation, thosedescribed in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al.,Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Masonet al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci.USA 83:3091-3095, 1986.

5.5.8. Chemotactic/Chemokinetic Activity

A protein of the present invention may have chemotactic or chemokineticactivity (e.g., act as a chemokine) for mammalian cells, including, forexample, monocytes, fibroblasts, neutrophils, T-cells, mast cells,eosinophils, epithelial and/or endothelial cells. A polynucleotide ofthe invention can encode a polypeptide exhibiting such attributes.Chemotactic and chemokinetic proteins can be used to mobilize or attracta desired cell population to a desired site of action. Chemotactic orchemokinetic proteins provide particular advantages in treatment ofwounds and other trauma to tissues, as well as in treatment of localizedinfections. For example, attraction of lymphocytes, monocytes orneutrophils to tumors or sites of infection may result in improvedimmune responses against the tumor or infecting agent.

A protein or peptide has chemotactic activity for a particular cellpopulation if it can stimulate, directly or indirectly, the directedorientation or movement of such cell population. Preferably, the proteinor peptide has the ability to directly stimulate directed movement ofcells. Whether a particular protein has chemotactic activity for apopulation of cells can be readily determined by employing such proteinor peptide in any known assay for cell chemotaxis.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for chemotactic activity (which will identify proteins thatinduce or prevent chemotaxis) consist of assays that measure the abilityof a protein to induce the migration of cells across a membrane as wellas the ability of a protein to induce the adhesion of one cellpopulation to another cell population. Suitable assays for movement andadhesion include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 6.12, Measurement of alpha and betaChemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol.25:1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnstonet al. J. of Immunol. 153:1762-1768, 1994.

5.5.9. Hemostatic and Thrombolytic Activity

A protein of the invention may also exhibit hemostatic or thrombolyticactivity. A polynucleotide of the invention can encode a polypeptideexhibiting such attributes. Such a protein is expected to be useful intreatment of various coagulation disorders (including hereditarydisorders, such as hemophilias) or to enhance coagulation and otherhemostatic events in treating wounds resulting from trauma, surgery orother causes. A protein of the invention may also be useful fordissolving or inhibiting formation of thromboses and for treatment andprevention of conditions resulting therefrom (such as, for example,infarction of cardiac and central nervous system vessels (e.g., stroke).

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assay for hemostatic and thrombolytic activity include, withoutlimitation, those described in: Linet et al., J. Clin. Pharmacol.26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987;Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins35:467-474, 1988.

5.5.10. Recentor/Ligand Activity

A protein of the present invention may also demonstrate activity asreceptors, receptor ligands or inhibitors or agonists of receptor/ligandinteractions. A polynucleotide of the invention can encode a polypeptideexhibiting such characteristics. Examples of such receptors and ligandsinclude, without limitation, cytokine receptors and their ligands,receptor kinases and their ligands, receptor phosphatases and theirligands, receptors involved in cell-cell interactions and their ligands(including without limitation, cellular adhesion molecules (such asselecting, integrins and their ligands) and receptor/ligand pairsinvolved in antigen presentation, antigen recognition and development ofcellular and humoral immune responses). Receptors and ligands are alsouseful for screening of potential peptide or small molecule inhibitorsof the relevant receptor/ligand interaction. A protein of the presentinvention (including, without limitation, fragments of receptors andligands) may themselves be useful as inhibitors of receptor/ligandinteractions.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for receptor-ligand activity include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28,Measurement of Cellular Adhesion under static conditions7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868,1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein etal., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol.Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

5.5.11. Anti-inflammatory Activity

Proteins of the present invention may also exhibit anti-inflammatoryactivity. The anti-inflammatory activity may be achieved by providing astimulus to cells involved in the inflammatory response, by inhibitingor promoting cell-cell interactions (such as, for example, celladhesion), by inhibiting or promoting chemotaxis of cells involved inthe inflammatory process, inhibiting or promoting cell extravasation, orby stimulating or suppressing production of other factors which moredirectly inhibit or promote an inflammatory response. Proteinsexhibiting such activities can be used to treat inflammatory conditionsincluding chronic or acute conditions), including without limitationintimation associated with infection (such as septic shock, sepsis orsystemic inflammatory response syndrome (SIRS)), ischemia-reperfusioninjury, endotoxin lethality, arthritis, complement-mediated hyperacuterejection, nephritis, cytokine or chemokine-induced lung injury,inflammatory bowel disease, Crohn's disease or resulting from overproduction of cytokines such as TNF or IL-1. Proteins of the inventionmay also be useful to treat anaphylaxis and hypersensitivity to anantigenic substance or material.

5.5.12. Leukemias

Leukemias and related disorders may be treated or prevented byadministration of a therapeutic that promotes or inhibits function ofthe polynucleotides and/or polypeptides of the invention. Such leukemiasand related disorders include but are not limited to acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic,promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronicleukemia, chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia (for a review of such disorders, see Fishman etal., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).

5.5.13. Nervous System Disorders

Nervous system disorders, involving cell types which can be tested forefficacy of intervention with compounds that modulate the activity ofthe polynucleotides and/or polypeptides of the invention, and which canbe treated upon thus observing an indication of therapeutic utility,include but are not limited to nervous system injuries, and diseases ordisorders which result in either a disconnection of axons, a diminutionor degeneration of neurons, or demyelination. Nervous system lesionswhich may be treated in a patient (including human and non-humanmammalian patients) according to the invention include but are notlimited to the following lesions of either the central (including spinalcord, brain) or peripheral nervous systems:

(i) traumatic lesions, including lesions caused by physical injury orassociated with surgery, for example, lesions which sever a portion ofthe nervous system, or compression injuries;

(ii) ischemic lesions, in which a lack of oxygen in a portion of thenervous system results in neuronal injury or death, including cerebralinfarction or ischemia, or spinal cord infarction or ischemia;

(iii) infectious lesions, in which a portion of the nervous system isdestroyed or injured as a result of infection, for example, by anabscess or associated with infection by human immunodeficiency virus,herpes zoster, or herpes simplex virus or with Lyme disease,tuberculosis, syphilis;

(iv) degenerative lesions, in which a portion of the nervous system isdestroyed or injured as a result of a degenerative process including butnot limited to degeneration associated with Parkinson's disease,Alzheimer's disease, Huntington's chorea, or amyotrophic lateralsclerosis;

(v) lesions associated with nutritional diseases or disorders, in whicha portion of the nervous system is destroyed or injured by a nutritionaldisorder or disorder of metabolism including but not limited to, vitaminB12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration;

(vi) neurological lesions associated with systemic diseases includingbut not limited to diabetes (diabetic neuropathy, Bell's palsy),systemic lupus erythematosus, carcinoma, or sarcoidosis;

(vii) lesions caused by toxic substances including alcohol, lead, orparticular neurotoxins; and

(viii) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including but notlimited to multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

Therapeutics which are useful according to the invention for treatmentof a nervous system disorder may be selected by testing for biologicalactivity in promoting the survival or differentiation of neurons. Forexample, and not by way of limitation, therapeutics which elicit any ofthe following effects may be useful according to the invention:

(i) increased survival time of neurons in culture;

(ii) increased sprouting of neurons in culture or in vivo;

(iii) increased production of a neuron-associated molecule in culture orin vivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or

(iv) decreased symptoms of neuron dysfunction in vivo.

Such effects may be measured by any method known in the art. Inpreferred, non-limiting embodiments, increased survival of neurons maybe measured by the method set forth in Arakawa et al. (1990, J.Neurosci. 10:3507-3515); increased sprouting of neurons may be detectedby methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) orBrown et al. (1981, Ann. Rev. Neurosci. 4:17-42); increased productionof neuron-associated molecules may be measured by bioassay, enzymaticassay, antibody binding, Northern blot assay, etc., depending on themolecule to be measured; and motor neuron dysfunction may be measured byassessing the physical manifestation of motor neuron disorder, e.g.,weakness, motor neuron conduction velocity, or functional disability.

In a specific embodiments, motor neuron disorders that may be treatedaccording to the invention include but are not limited to disorders suchas infarction, infection, exposure to toxin, trauma, surgical damage,degenerative disease or malignancy that may affect motor neurons as wellas other components of the nervous system, as well as disorders thatselectively affect neurons such as amyotrophic lateral sclerosis, andincluding but not limited to progressive spinal muscular atrophy,progressive bulbar palsy, primary lateral sclerosis, infantile andjuvenile muscular atrophy, progressive bulbar paralysis of childhood(Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, andHereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

5.5.14. Other Activities

A protein of the invention may also exhibit one or more of the followingadditional activities or effects: inhibiting the growth, infection orfunction of, or killing, infectious agents, including, withoutlimitation, bacteria, viruses, fungi and other parasites; effecting(suppressing or enhancing) bodily characteristics, including, withoutlimitation, height, weight, hair color, eye color, skin, fat to leanratio or other tissue pigmentation, or organ or body part size or shape(such as, for example, breast augmentation or diminution, change in boneform or shape); effecting biorhythms or caricadic cycles or rhythms;effecting the fertility of male or female subjects; effecting themetabolism, catabolism, anabolism, processing, utilization, storage orelimination of dietary fat, lipid, protein, carbohydrate, vitamins,minerals, co-factors or other nutritional factors or component(s);effecting behavioral characteristics, including, without limitation,appetite, libido, stress, cognition (including cognitive disorders),depression (including depressive disorders) and violent behaviors;providing analgesic effects or other pain reducing effects; promotingdifferentiation and growth of embryonic stem cells in lineages otherthan hematopoietic lineages; hormonal or endocrine activity; in the caseof enzymes, correcting deficiencies of the enzyme and treatingdeficiency-related diseases; treatment of hyperproliferative disorders(such as, for example, psoriasis); immunoglobulin-like activity (suchas, for example, the ability to bind antigens or complement); and theability to act as an antigen in a vaccine composition to raise an immuneresponse against such protein or another material or entity which iscross-reactive with such protein.

5.6. Pharmaceutical Formulations and Routes of Administration

A protein of the present invention (from whatever source derived,including without limitation from recombinant and non-recombinantsources) may be administered to a patient in need, by itself, or inpharmaceutical compositions where it is mixed with suitable carriers orexcipient(s) at doses to treat or ameliorate a variety of disorders.Such a composition may also contain (in addition to protein and acarrier) diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials well known in the art. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The characteristics of the carrier will depend on the route ofadministration. The pharmaceutical composition of the invention may alsocontain cytokines, lymphokines, or other hematopoietic factors such asM-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2,G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.The pharmaceutical composition may further contain other agents whicheither enhance the activity of the protein or compliment its activity oruse in treatment. Such additional factors and/or agents may be includedin the pharmaceutical composition to produce a synergistic effect withprotein of the invention, or to minimize side effects. Conversely,protein of the present invention may be included in formulations of theparticular cytokine, lymphokine, other hematopoietic factor,thrombolytic or anti-thrombotic factor, or anti-inflammatory agent tominimize side effects of the cytokine, lymphokine, other hematopoieticfactor, thrombolytic or anti-thrombotic factor, or anti-inflammatoryagent. A protein of the present invention may be active in multimers(e.g., heterodimers or homodimers) or complexes with itself or otherproteins. As a result, pharmaceutical compositions of the invention maycomprise a protein of the in invention in such multimeric or complexedform.

Techniques for formulation and administration of the compounds of theinstant application may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition. Atherapeutically effective dose further refers to that amount of thecompound sufficient to result in amelioration of symptoms, e.g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of protein of the present invention isadministered to a mammal having a condition to be treated. Protein ofthe present invention may be administered in accordance with the methodof the invention either alone or in combination with other therapiessuch as treatments employing cytokines, lymphokines or otherhematopoietic factors. When co-administered with one or more cytokines,lymphokines or other hematopoietic factors, protein of the presentinvention may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering protein of the present invention incombination with cytokine(s), lymphokine(s), other hematopoieticfactor(s), thrombolytic or anti-thrombotic factors.

5.6.1. Routes of Administration

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. Administrationof protein of the present invention used in the pharmaceuticalcomposition or to practice the method of the present invention can becarried out in a variety of conventional ways, such as oral ingestion,inhalation, topical application or cutaneous, subcutaneous,intraperitoneal, parenteral or intravenous injection. Intravenousadministration to the patient is preferred.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a arthritic joints or in fibrotic tissue, often in a depot orsustained release formulation. In order to prevent the scarring processfrequently occurring as complication of glaucoma surgery, the compoundsmay be administered topically, for example, as eye drops. Furthermore,one may administer the drug in a targeted drug delivery system, forexample, in a liposome coated with a specific antibody, targeting, forexample, arthritic or fibrotic tissue. The liposomes will be targeted toand taken up selectively by the afflicted tissue.

5.6.2. Compositions/Formulations

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. These pharmaceuticalcompositions may be manufactured in a manner in that is itself known,e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes. Proper formulation is dependent upon the routeof administration chosen. When a therapeutically effective amount ofprotein of the present invention is administered orally, protein of thepresent invention will be in the form of a tablet, capsule, powder,solution or elixir. When administered in tablet form, the pharmaceuticalcomposition of the invention may additionally contain a solid carriersuch as a gelatin or an adjuvant. The tablet, capsule, and powdercontain from about 5 to 95% protein of the present invention, andpreferably from about 25 to 90% protein of the present invention. Whenadministered in liquid form, a liquid carrier such as water, petroleum,oils of animal or plant origin such as peanut oil, mineral oil, soybeanoil, or sesame oil, or synthetic oils may be added. The liquid form ofthe pharmaceutical composition may further contain physiological salinesolution, dextrose or other saccharide solution, or glycols such asethylene glycol, propylene glycol or polyethylene glycol. Whenadministered in liquid form, the pharmaceutical composition containsfrom about 0.5 to 90% by weight of protein of the present invention, andpreferably from about 1 to 50% protein of the present invention.

When a therapeutically effective amount of protein of the presentinvention is administered by intravenous, cutaneous or subcutaneousinjection, protein of the present invention will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection should contain, in addition to protein of thepresent invention, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art. Forinjection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. The compounds maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, orsynthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, the compounds may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. The cosolventsystem may be the VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5%dextrose in water solution. This co-solvent system dissolves hydrophobiccompounds well, and itself produces low toxicity upon systemicadministration. Naturally, the proportions of a co-solvent system may bevaried considerably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose. Alternatively, otherdelivery systems for hydrophobic pharmaceutical compounds may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also may be employed, although usually at thecost of greater toxicity. Additionally, the compounds may be deliveredusing a sustained-release system, such as semipermeable matrices ofsolid hydrophobic polymers containing the therapeutic agent. Various ofsustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the compounds for a few weeks up to over100 days. Depending on the chemical nature and the biological stabilityof the therapeutic reagent, additional strategies for proteinstabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols. Many of the proteinase inhibitingcompounds of the invention may be provided as salts withpharmaceutically compatible counterions. Such pharmaceuticallyacceptable base addition salts are those salts which retain thebiological effectiveness and properties of the free acids and which areobtained by reaction with inorganic or organic bases such as sodiumhydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine,monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate,triethanol amine and the like.

The pharmaceutical composition of the invention may be in the form of acomplex of the protein(s) of present invention along with protein orpeptide antigens. The protein and/or peptide antigen will deliver astimulatory signal to both B and T lymphocytes. B lymphocytes willrespond to antigen through their surface immunoglobulin receptor. Tlymphocytes will respond to antigen through the T cell receptor (TCR)following presentation of the antigen by MHC proteins. MHC andstructurally related proteins including those encoded by class I andclass II MHC genes on host cells will serve to present the peptideantigen(s) to T lymphocytes; The antigen components could also besupplied as purified MHC-peptide complexes alone or with co-stimulatorymolecules that can directly signal T cells. Alternatively antibodiesable to bind surface immunoglobulin and other molecules on B cells aswell as antibodies able to bind the TCR and other molecules on T cellscan be combined with the pharmaceutical composition of the invention.The pharmaceutical composition of the invention may be in the form of aliposome in which protein of the present invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728;4,837,028; and 4,737,323, all of which are incorporated herein byreference.

The amount of protein of the present invention in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of protein of the present inventionwith which to treat each individual patient. Initially, the attendingphysician will administer low doses of protein of the present inventionand observe the patient's response. Larger doses of protein of thepresent invention may be administered until the optimal therapeuticeffect is obtained for the patient, and at that point the dosage is notincreased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention shouldcontain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about10 mg, more preferably about 0.1 μg to about 1 mg) of protein of thepresent invention per kg body weight. For compositions of the presentinvention which are useful for bone, cartilage, tendon or ligamentregeneration, the therapeutic method includes administering thecomposition topically, systematically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Further, the composition may desirably be encapsulated or injectedin a viscous form for delivery to the site of bone, cartilage or tissuedamage. Topical administration may be suitable for wound healing andtissue repair. Therapeutically useful agents other than a protein of theinvention which may also optionally be included in the composition asdescribed above, may alternatively or additionally, be administeredsimultaneously or sequentially with the composition in the methods ofthe invention. Preferably for bone and/or cartilage formation, thecomposition would include a matrix capable of delivering theprotein-containing composition to the site of bone and/or cartilagedamage, providing a structure for the developing bone and cartilage andoptimally capable of being resorbed into the body. Such matrices may beformed of materials presently in use for other implanted medicalapplications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalciumphosphate. The bioceramics may be altered in composition, suchas in calcium-aluminate-phosphate and processing to alter pore size,particle size, particle shape, and biodegradability. Presently preferredis a 50:50 (mole weight) copolymer of lactic acid and glycolic acid inthe form of porous particles having diameters ranging from 150 to 800microns. In some applications, it will be useful to utilize asequestering agent, such as carboxymethyl cellulose or autologous bloodclot, to prevent the protein compositions from disassociating from thematrix.

A preferred family of sequestering agents is cellulosic materials suchas alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropyl-methylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorbtion of the protein from the polymer matrixand to provide appropriate handling of the composition, yet not so muchthat the progenitor cells are prevented from infiltrating the matrix,thereby providing the protein the opportunity to assist the osteogenicactivity of the progenitor cells. In further compositions, proteins ofthe invention may be combined with other agents beneficial to thetreatment of the bone and/or cartilage defect, wound, or tissue inquestion. These agents include various growth factors such as epidermalgrowth factor (EGF), platelet derived growth factor (PDGF), transforminggrowth factors (TGF-.alpha. and TGF-.beta.), and insulin-like growthfactor (IGF).

The therapeutic compositions are also presently valuable for veterinaryapplications. Particularly domestic animals and thoroughbred horses, inaddition to humans, are desired patients for such treatment withproteins of the present invention. The dosage regimen of aprotein-containing pharmaceutical composition to be used in tissueregeneration will be determined by the attending physician consideringvarious factors which modify the action of the proteins, e.g., amount oftissue weight desired to be formed, the site of damage, the condition ofthe damaged tissue, the size of a wound, type of damaged tissue (e.g.,bone), the patient's age, sex, and diet, the severity of any infection,time of administration and other clinical factors. The dosage may varywith the type of matrix used in the reconstitution and with inclusion ofother proteins in the pharmaceutical composition. For example, theaddition of other known growth factors, such as IGF I (insulin likegrowth factor I), to the final composition, may also effect the dosage.Progress can be monitored by periodic assessment of tissue/bone growthand/or repair, for example, X-rays, histomorphometric determinations andtetracycline labeling.

Polynucleotides of the present invention can also be used for genetherapy. Such polynucleotides can be introduced either in vivo or exvivo into cells for expression in a mammalian subject. Polynucleotidesof the invention may also be administered by other known methods forintroduction of nucleic acid into a cell or organism (including, withoutlimitation, in the form of viral vectors or naked DNA). Cells may alsobe cultured ex vivo in the presence of proteins of the present inventionin order to proliferate or to produce a desired effect on or activity insuch cells. Treated cells can then be introduced in vivo for therapeuticpurposes.

5.6.3. Effective Dosage

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amounts is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein. For any compound used in the methodof the invention, the therapeutically effective dose can be estimatedinitially from cell culture assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of the C-proteinase activity). Such information can be usedto more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms or a prolongation of survivalin a patient. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds which exhibit high therapeutic indicesare preferred. The data obtained from these cell culture assays andanimal studies can be used in formulating a range of dosage for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p.1. Dosage amount and interval may be adjusted individually toprovide plasma levels of the active moiety which are sufficient tomaintain the C-proteinase inhibiting effects, or minimal effectiveconcentration (MEC). The MEC will vary for each compound but can beestimated from in vitro data; for example, the concentration necessaryto achieve 50-90% inhibition of the C-proteinase using the assaysdescribed herein. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. However, HPLCassays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%. In cases of local administration or selectiveuptake, the effective local concentration of the drug may not be relatedto plasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

5.6.4. Packaging

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may, for example, comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabelled for treatment of an indicated condition.

5.7. Antibodies

Another aspect of the invention is an antibody that specifically bindsthe polypeptide of the invention. Such antibodies can be eithermonoclonal or polyclonal antibodies, as well fragments thereof andhumanized forms or fully human forms, such as those produced intransgenic animals. The invention further provides a hybridoma thatproduces an antibody according to the invention. Antibodies of theinvention are useful for detection and/or purification of thepolypeptides of the invention.

Protein of the invention may also be used to immunize animals to obtainpolyclonal and monoclonal antibodies which specifically react with theprotein. Such antibodies may be obtained using either the entire proteinor fragments thereof as an immunogen. The peptide immunogensadditionally may contain a cysteine residue at the carboxyl terminus,and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).Methods for synthesizing such peptides are known in the art, forexample, as in R. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154(1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987). Monoclonalantibodies binding to the protein of the invention may be usefuldiagnostic agents for the immunodetection of the protein. Neutralizingmonoclonal antibodies binding to the protein may also be usefultherapeutics for both conditions associated with the protein and also inthe treatment of some forms of cancer where abnormal expression of theprotein is involved. In the case of cancerous cells or leukemic cells,neutralizing monoclonal antibodies against the protein may be useful indetecting and preventing the metastatic spread of the cancerous cells,which may be mediated by the protein. In general, techniques forpreparing polyclonal and monoclonal antibodies as well as hybridomascapable of producing the desired antibody are well known in the art(Campbell, A. M., Monoclonal Antibodies Technology: LaboratoryTechniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers, Amsterdam, The Netherlands (1984); St. Groth et al., J.Immunol. 35:1-21 (1990); Kohler and Milstein, Nature 256:495-497(1975)), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., Immunology Today 4:72 (1983); Cole et al., in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), pp. 77-96).

Any animal (mouse, rabbit, etc.) which is known to produce antibodiescan be immunized with a peptide or polypeptide of the invention. Methodsfor immunization are well known in the art. Such methods includesubcutaneous or intraperitoneal injection of the polypeptide. Oneskilled in the art will recognize that the amount of the protein encodedby the ORF of the present invention used for immunization will varybased on the animal which is immunized, the antigenicity of the peptideand the site of injection. The protein that is used as an immunogen maybe modified or administered in an adjuvant in order to increase theprotein's antigenicity. Methods of increasing the antigenicity of aprotein are well known in the art and include, but are not limited to,coupling the antigen with a heterologous protein (such as globulin orβ-galactosidase) or through the inclusion of an adjuvant duringimmunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, andallowed to become monoclonal antibody producing hybridoma cells. Any oneof a number of methods well known in the art can be used to identify thehybridoma cell which produces an antibody with the desiredcharacteristics. These include screening the hybridomas with an ELISAassay, western blot analysis, or radioimmunoassay (Lutz et al., Exp.Cell Research. 175:109-124 (1988)). Hybridomas secreting the desiredantibodies are cloned and the class and subclass is determined usingprocedures known in the art (Campbell, A. M., Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and Molecular Biology,Elsevier Science Publishers, Amsterdam, The Netherlands (1984)).Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toproteins of the present invention.

For polyclonal antibodies, antibody containing antiserum is isolatedfrom the immunized animal and is screened for the presence of antibodieswith the desired specificity using one of the above-describedprocedures. The present invention further provides the above-describedantibodies in delectably labeled form. Antibodies can be delectablylabeled through the use of radioisotopes, affinity labels (such asbiotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase,alkaline phosphatase, etc.) fluorescent labels (such as FITC orrhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishingsuch labeling are well-known in the art, for example, see (Sternberger,L. A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. etal., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129(1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

The labeled antibodies of the present invention can be used for invitro, in vivo, and in situ assays to identify cells or tissues in whicha fragment of the polypeptide of interest is expressed. The antibodiesmay also be used directly in therapies or other diagnostics. The presentinvention further provides the above-described antibodies immobilized ona solid support. Examples of such solid supports include plastics suchas polycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir, D. M. et al., “Handbook of Experimental Immunology” 4th Ed.,Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986);Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N.Y. (1974)). Theimmobilized antibodies of the present invention can be used for invitro, in vivo, and in situ assays as well as for immuno-affinitypurification of the proteins of the present invention.

5.8. Computer Readable Sequences

In one application of this embodiment, a nucleotide sequence of thepresent invention can be recorded on computer readable media. As usedherein, “computer readable media” refers to any medium which can be readand accessed directly by a computer. Such media include, but are notlimited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemediums can be used to create a manufacture comprising computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention. As used herein, “recorded” refers to a process for storinginformation on computer readable medium. A skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate manufactures comprising thenucleotide sequence information of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide sequence of the present invention. The choice of the datastorage structure will generally be based on the means chosen to accessthe stored information. In addition, a variety of data processorprograms and formats can be used to store the nucleotide sequenceinformation of the present invention on computer readable medium. Thesequence information can be represented in a word processing text file,formatted in commercially-available software such as WordPerfect andMicrosoft Word, or represented in the form of an ASCII file, stored in adatabase application, such as DB2, Sybase, Oracle, or the like. Askilled artisan can readily adapt any number of dataprocessorstructuring formats (e.g. text file or database) in order to obtaincomputer readable medium having recorded thereon the nucleotide sequenceinformation of the present invention. By providing the nucleotidesequence of SEQ ID NO:1 or a representative fragment thereof, or anucleotide sequence at least 99.9% identical to SEQ ID NO:1in computerreadable form, a skilled artisan can routinely access the sequenceinformation for a variety of purposes. Computer software is publiclyavailable which allows a skilled artisan to access sequence informationprovided in a computer readable medium. The examples which followdemonstrate how software which implements the BLAST (Altschul et al., J.Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem.17:203-207 (1993)) search algorithms on a Sybase system is used toidentify open reading frames (ORFs) within a nucleic acid sequence. SuchORFs may be protein encoding fragments and may be useful in producingcommercially important proteins such as enzymes used in fermentationreactions and in the production of commercially useful metabolites.

As used herein, “a computer-based system” refers to the hardware means,software means, and data storage means used to analyze the nucleotidesequence information of the present invention. The minimum hardwaremeans of the computer-based systems of the present invention comprises acentral processing unit (CPU), input means, output means, and datastorage means. A skilled artisan can readily appreciate that any one ofthe currently available computer-based systems are suitable for use inthe present invention. As stated above, the computer-based systems ofthe present invention comprise a data storage means having storedtherein a nucleotide sequence of the present invention and the necessaryhardware means and software means for supporting and implementing asearch means. As used herein, “data storage means” refers to memorywhich can store nucleotide sequence information of the presentinvention, or a memory access means which can access manufactures havingrecorded thereon the nucleotide sequence information of the presentinvention.

As used herein, “search means” refers to one or more programs which areimplemented on the computer-based system to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of a known sequence which match a particular target sequence ortarget motif. A variety of known algorithms are disclosed publicly and avariety of commercially available software for conducting search meansare and can be used in the computer-based systems of the presentinvention. Examples of such software includes, but is not limited to,MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A skilled artisancan readily recognize that any one of the available algorithms orimplementing software packages for conducting homology searches can beadapted for use in the present computer-based systems. As used herein, a“target sequence” can be any nucleic acid or amino acid sequence of sixor more nucleotides or two or more amino acids. A skilled artisan canreadily recognize that the longer a target sequence is, the less likelya target sequence will be present as a random occurrence in thedatabase. The most preferred sequence length of a target sequence isfrom about 10 to 100 amino acids or from about 30 to 300 nucleotideresidues. However, it is well recognized that searches for commerciallyimportant fragments, such as sequence fragments involved in geneexpression and protein processing, may be of shorter length.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen based on a three-dimensional configurationwhich is formed upon the folding of the target motif. There are avariety of target motifs known in the art. Protein target motifsinclude, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein binding sequences).

5.9. Triplex Helix Formation

In addition, the fragments of the present invention, as broadlydescribed, can be used to control gene expression through triple helixformation or antisense DNA or RNA, both of which methods are based onthe binding of a polynucleotide sequence to DNA or RNA. Polynucleotidessuitable for use in these methods are usually 20 to 40 bases in lengthand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 15241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triplehelix-formation optimally results in a shut-off of RNA transcriptionfrom DNA, while antisense RNA hybridization blocks translation of anmRNA molecule into polypeptide. Both techniques have been demonstratedto be effective in model systems. Information contained in the sequencesof the present invention is necessary for the design of an antisense ortriple helix oligonucleotide.

5.10. Diagnostic Assays and Kits

The present invention further provides methods to identify the presenceor expression of one of the ORFs of the present invention, or homologthereof, in a test sample, using a nucleic acid probe or antibodies ofthe present invention.

In general, methods for detecting a polynucleotide of the invention cancomprise contacting a'sample with a compound that binds to and forms acomplex with the polynucleotide for a period sufficient to form thecomplex, and detecting the complex, so that if a complex is detected, apolynucleotide of the invention is detected in the sample.

Such methods can also comprise contacting a sample under stringenthybridization conditions with nucleic acid primers that anneal to apolynucleotide of the invention under such conditions, and amplifyingannealed polynucleotides, so that if a polynucleotide is amplified, apolynucleotide of the invention is detected in the sample.

In general, methods for detecting a polypeptide of the invention cancomprise contacting a sample with a compound that binds to and forms acomplex with the polypeptide for a period sufficient to form thecomplex, and detecting the complex, so that if a complex is detected, apolypeptide of the invention is detected in the sample.

In detail, such methods comprise incubating a test sample with one ormore of the antibodies or one or more of nucleic acid probes of thepresent invention and assaying for binding of the nucleic acid probes orantibodies to components within the test sample.

Conditions for incubating a nucleic acid probe or antibody with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid probe or antibody used in the assay. One skilled in the artwill recognize that any one of the commonly available hybridization,amplification or immunological assay formats can readily be adapted toemploy the nucleic acid probes or antibodies of the present invention.Examples of such assays can be found in Chard, T., An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985). The test samplesof the present invention include cells, protein or membrane extracts ofcells, or biological fluids such as sputum, blood, serum, plasma, orurine. The test sample used in the above-described method will varybased on the assay format, nature of the detection method and thetissues, cells or extracts used as the sample to be assayed. Methods forpreparing protein extracts or membrane extracts of cells are well knownin the art and can be readily be adapted in order to obtain a samplewhich is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention. Specifically, the invention provides a compartment kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the probes or antibodies of thepresent invention; and (b) one or more other containers comprising oneor more of the following: wash reagents, reagents capable of detectingpresence of a bound probe or antibody.

In detail, a compartment kit includes any kit in which reagents arecontained in separate containers. Such containers include small glasscontainers, plastic containers or strips of plastic or paper. Suchcontainers allows one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers will include a container which will accept thetest sample, a container which contains the antibodies used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, etc.), and containers which contain thereagents used to detect the bound antibody or probe. Types of detectionreagents include labeled nucleic acid probes, labeled secondaryantibodies, or in the alternative, if the primary antibody is labeled,the enzymatic, or antibody binding reagents which are capable ofreacting with the labeled antibody. One skilled in the art will readilyrecognize that the disclosed probes and antibodies of the presentinvention can be readily incorporated into one of the established kitformats which are well known in the art.

5.11. Screening Assays

Using the isolated proteins and polynucleotides of the invention, thepresent invention further provides methods of obtaining and identifyingagents which bind to a protein encoded by the ORF from a nucleic acidwith a sequence of SEQ ID NO:1, to a specific domain of the polypeptideencoded by the nucleic acid, or to a nucleic acid with a sequence of SEQID NO:1. In detail, said method comprises the steps of:

(a) contacting an agent with an isolated protein encoded by an ORF ofthe present invention, or nucleic acid of the invention; and

(b) determining whether the agent binds to said protein or said nucleicacid.

In general, therefore, such methods for identifying compounds that bindto a polynucleotide of the invention can comprise contacting a compoundwith a polynucleotide of the invention for a time sufficient to form apolynucleotide/compound complex, and detecting the complex, so that if apolynucleotide/compound complex is detected, a compound that binds to apolynucleotide of the invention is identified.

Likewise, in general, therefore, such methods for identifying compoundsthat bind to a polypeptide of the invention can comprise contacting acompound with a polypeptide of the invention for a time sufficient toform a polypeptide/compound complex, and detecting the complex, so thatif a polypeptide/compound complex is detected, a compound that binds toa polynucleotide of the invention is identified.

Methods for identifying compounds that bind to a polypeptide of theinvention can also comprise contacting a compound with a polypeptide ofthe invention in a cell for a time sufficient to form apolypeptide/compound complex, wherein the complex drives expression of areceptor gene sequence in the cell, and detecting the complex bydetecting reporter gene sequence expression, so that if apolypeptide/compound complex is detected, a compound that binds apolypeptide of the invention is identified.

Compounds identified via such methods can include compounds whichmodulate the activity of a polypeptide of the invention (that is,increase or decrease its activity, relative to activity observed in theabsence of the compound). Alternatively, compounds identified via suchmethods can include compounds which modulate the expression of apolynucleotide of the invention (that is, increase or decreaseexpression relative to expression levels observed in the absence of thecompound). Compounds, such as compounds identified via the methods ofthe invention, can be tested using standard assays well known to thoseof skill in the art for their ability to modulate activity/expression.

The agents screened in the above assay can be, but are not limited to,peptides, carbohydrates, vitamin derivatives, or other pharmaceuticalagents. The agents can be selected and screened at random or rationallyselected or designed using protein modeling techniques.

For random screening, agents such as peptides, carbohydrates,pharmaceutical agents and the like are selected at random and areassayed for their ability to bind to the protein encoded by the ORF ofthe present invention. Alternatively, agents may be rationally selectedor designed. As used herein, an agent is said to be “rationally selectedor designed” when the agent is chosen based on the configuration of theparticular protein. For example, one skilled in the art can readilyadapt currently available procedures to generate peptides,pharmaceutical agents and the like capable of binding to a specificpeptide sequence in order to generate rationally designed antipeptidepeptides, for example see Hurby et al., Application of SyntheticPeptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide,W.H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry28:9230-8 (1989), or pharmaceutical agents, or the like.

In addition to the foregoing, one class of agents of the presentinvention, as broadly described, can be used to control gene expressionthrough binding to one of the ORFs or EMFs of the present invention. Asdescribed above, such agents can be randomly screened or rationallydesigned/selected. Targeting the ORF or EMF allows a skilled artisan todesign sequence specific or element specific agents, modulating theexpression of either a single ORF or multiple ORFs which rely on thesame EMF for expression control. One class of DNA binding agents areagents which contain base residues which hybridize or form a triplehelix formation by binding to DNA or RNA. Such agents can be based onthe classic phosphodiester, ribonucleic acid backbone, or can be avariety of sulfhydryl or polymeric derivatives which have baseattachment capacity.

Agents suitable for use in these methods usually contain 20 to 40 basesand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triplehelix-formation optimally results in a shut-off of RNA transcriptionfrom DNA, while antisense RNA hybridization blocks translation of anmRNA molecule into polypeptide. Both techniques have been demonstratedto be effective in model systems. Information contained in the sequencesof the present invention is necessary for the design of an antisense ortriple helix oligonucleotide and other DNA binding agents. Agents whichbind to a protein encoded by one of the ORFs of the present inventioncan be used as a diagnostic agent, in the control of bacterial infectionby modulating the activity of the protein encoded by the ORF. Agentswhich bind to a protein encoded by one of the ORFs of the presentinvention can be formulated using known techniques to generate apharmaceutical composition.

5.10. Use of Nucleic Acids as Probes

Another aspect of the subject invention is to provide forpolypeptide-specific nucleic acid hybridization probes capable ofhybridizing with naturally occurring nucleotide sequences. Thehybridization probes of the subject invention may be derived from thenucleotide sequence of the SEQ ID NO:1. Because the corresponding geneis only expressed in a limited number of tissues, especially adulttissues, a hybridization probe derived from SEQ ID NO:1 can be used asan indicator of the presence of RNA of cell type of such a tissue in asample.

Any suitable hybridization technique can be employed, such as, forexample, in situ hybridization. PCR as described U.S. Pat. Nos.4,683,195 and 4,965,188 provides additional uses for oligonucleotidesbased upon the nucleotide sequences. Such probes used in PCR may be ofrecombinant origin, may be chemically synthesized, or a mixture of both.The probe will comprise a discrete nucleotide sequence for the detectionof identical sequences or a degenerate pool of possible sequences foridentification of closely related genomic sequences.

Other means for producing specific hybridization probes for nucleicacids include the cloning of nucleic acid sequences into vectors for theproduction of mRNA probes. Such vectors are known in the art and arecommercially available and may be used to synthesize RNA probes in vitroby means of the addition of the appropriate RNA polymerase as T7 or SP6RNA polymerase and the appropriate radioactively labeled nucleotides.The nucleotide sequences may be used to construct hybridization probesfor mapping their respective genomic sequences. The nucleotide sequenceprovided herein may be mapped to a chromosome or specific regions of achromosome using well known genetic and/or chromosomal mappingtechniques. These techniques include in situ hybridization, linkageanalysis against known chromosomal markers, hybridization screening withlibraries or flow-sorted chromosomal preparations specific to knownchromosomes, and the like. The technique of fluorescent in situhybridization of chromosome spreads has been described, among otherplaces, in Verma et al (1988) Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York N.Y.

Fluorescent in situ hybridization of chromosomal preparations and otherphysical chromosome mapping techniques may be correlated with additionalgenetic map data. Examples of genetic map data can be found in the 1994Genome Issue of Science (265:1981f). Correlation between the location ofa nucleic acid on a physical chromosomal map and a specific disease (orpredisposition to a specific disease) may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier or affected individuals. The nucleotide sequencemay be used to produce purified polypeptides using well known methods ofrecombinant DNA technology. Among the many publications that teachmethods for the expression of genes after they have been isolated isGoeddel (1990) Gene Expression Technology, Methods and Enzymology, Vol185, Academic Press, San Diego. Polypeptides may be expressed in avariety of host cells, either prokaryotic or eukaryotic. Host cells maybe from the same species from which a particular polypeptide nucleotidesequence was isolated or from a different species. Advantages ofproducing polypeptides by recombinant DNA technology include obtainingadequate amounts of the protein for purification and the availability ofsimplified purification procedures.

Each sequence so obtained was compared to sequences in GenBank using asearch algorithm developed by Applied Biosystems and incorporated intothe INHERIT™ 670 Sequence Analysis System. In this algorithm, PatternSpecification Language (developed by TRW Inc., Los Angeles, Calif.) wasused to determine regions of homology. The three parameters thatdetermine how the sequence comparisons run were window size, windowoffset, and error tolerance. Using a combination of these threeparameters, the DNA database was searched for sequences containingregions of homology to the query sequence, and the appropriate sequenceswere scored with an initial value. Subsequently, these homologousregions were examined using dot matrix homology plots to distinguishregions of homology from chance matches. Smith-Waterman alignments wereused to display the results of the homology search. Peptide and proteinsequence homologies were ascertained using the INHERIT™ 670 SequenceAnalysis System in a way similar to that used in DNA sequencehomologies. Pattern Specification Language and parameter windows wereused to search protein databases for sequences containing regions ofhomology that were scored with an initial value. Dot-matrix homologyplots were examined to distinguish regions of significant homology fromchance matches.

Alternatively, BLAST, which stands for Basic Local Alignment SearchTool, is used to search for local sequence alignments (Altschul S F(1993) J Mol Evol 36:290-300; Altschul, S F et al (1990) J Mol Biol215:403-10). BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. Whereas it is ideal for matcheswhich do not contain gaps, it is inappropriate for performingmotif-style searching. The fundamental unit of BLAST algorithm output isthe High-scoring Segment Pair (HSP). An HSP consists of two sequencefragments of arbitrary but equal lengths whose alignment is locallymaximal and for which the alignment score meets or exceeds a thresholdor cutoff score set by the user. The BLAST approach is to look for HSPsbetween a query sequence and a database sequence, to evaluate thestatistical significance of any matches found, and to report only thosematches which satisfy the user-selected threshold of significance. Theparameter E establishes the statistically significant threshold forreporting database sequence matches. E is interpreted as the upper boundof the expected frequency of chance occurrence of an HSP (or set ofHSPs) within the context of the entire database search. Any databasesequence whose match satisfies E is reported in the program output.

In addition, BLAST analysis was used to search for related moleculeswithin the libraries of the LIFESEQ™ database. This process, an“electronic northern” analysis is analogous to northern blot analysis inthat it uses one cellubrevin sequence at a time to search for identicalor homologous molecules at a set stringency. The stringency of theelectronic northern is based on “product score”. The product score isdefined as (% nucleotide or amino acid [between the query and referencesequences] in Blast multiplied by the % maximum possible BLAST score[based on the lengths of query and reference sequences]) divided by 100.At a product score of 40, the match will be exact within a 1-2% error;and at 70, the match will be exact. Homologous or related molecules canbe identified by selecting those which show product scores betweenapproximately 15 and 30.

The present invention is illustrated in the following examples. Uponconsideration of the present disclosure, one of skill in the art willappreciate that many other embodiments and variations may be made in thescope of the present invention. Accordingly, it is intended that thebroader aspects of the present invention not be limited to thedisclosure of the following examples.

6. EXAMPLE Identification of Novel CD-39-like Nucleic Acid andPolynucleotide Molecules

Described herein is the cloning and characterization of novel CD-39-likenucleotide-triphosphatase (“NTPase”) gene and polypeptide sequences.These sequences are referred to below as “mNTPase”, “mCD39L4” and“CD39L2.”

First, a novel murine family member was cloned by low stringencyscreening of mouse cDNA libraries with a human CD39L1 cDNA clone(Chadwick, B. P. & Frischauf A.-M., 1997, Mamm. Genome 8:668672). A 1738bp cDNA clone was isolated from an adult mouse testis cDNA library(Stratagene Ltd., Cambridge, UK) and sequenced. DNA sequence comparisonswith the human CD39L1cDNA sequence showed moderate DNA homology ofapproximately 39% identity). An open reading frame (ORF) could bedetected for the cNDA sequence, but indicated that the cDNA clone didnot contain the initiation methionine codon and, therefore, did notextend to the 5′ end. Database searches with the mouse cDNA sequenceidentified two mouse EST clones that extended the cDNA sequence at the5′ end (Accession Nos. AA116990 and AA120757). The EST clones wereresequenced. The cloned and the resequenced nucleotide sequences wereanalyzed and were combined appropriately to yield the nucleotidesequence (SEQ ID NO:7) depicted in FIGS. 1A, 1B, 1C, 1D, 1E, and 1F, andreferred to herein as mCD39L4 or mNTPase. The sequence revealed an ORFfrom nucleotides 205 to 1599 with the ATG at nucleotide 205 having amoderate match to the initiation start site for vertebrates (AAGAAUAUGG(SEO ID NO: 18) for mNTPase versus GCCGCCAUGG (SEQ ID NO: 19); Kozak,M., 1989, J. Biol. Chem. 108:229-241). The derived amino acid sequenceis also shown in FIGS. 2A, 2B, and 2C (SEQ ID NO:8). No apparentpolyadenylation signal existed, although the cDNA clone isolatedcontained a poly-A tail.

Hydropathy plots with Topred-II 1.1 (Claros, M. G. & Von Hejine, G.,1994, Comput. Appl. Biosci. 10:685-686) predict a single potentialtransmembrane segment close to the amino terminus of the protein,suggesting a single-pass transmembrane protein with a largeextracellular domain. Two potential glycosylation sites can be found atamino acid positions 41 (NVSA) and at 231 (NSTF), suggesting thatmNTPase is glycosylated.

Database searches with the derived amino acid sequence identifiedhomology with other members of the NTPase family. FIGS. 2A, 2B, and 2Cshow an alignment of the full mNTPase (mCD39L4) protein sequence againstthree of the most homologous known NTPases, from garden pea, potato andSaccharomyces cerevisiae. The mNTPase protein shares approximately 30%amino acid identity with the three other NTPases.

The region of highest homology between all members of the NTPase familyis at the amino terminus of the protein. Handa & Guidotti (Handa, M. &Guidotti, G., 1996, Biochem. Biophys. Res. Commun. 218:916-923)highlighted four regions of NTPases referred to as putativeapyrase-conserved regions (“ACRs”). FIGS. 3A, 3B, 3C, and 3D show analignment of ACRs I-IV. (See Section 3, Section 5, and its subsections,above, for a delineation of the amino acid residues that make up ACRsI-IV of the CD39-like polypeptides of the invention.) ACR conservationwould indicate that these regions are essential for the functioning ofthe protein, while changes in the regions surrounding these domains canbe tolerated. The presence of all four ACRs in the mNTPase (mCD39L2)indicates that mNTPase is a new member of the NTPase family.

BLAST searches with the DNA sequence of mNTPase (mCD39L4) revealed twooverlapping human EST clones with 57% DNA sequence identity to portionsof mNTPase (Accession Nos. H08436 and AA378537). Upon combination andanalysis of the resulting sequence, an ORF was identified that showedhomology to NTPases. The putative NTPase protein sequence, referred toherein as “CD39L2,” is shown in FIGS. 3A, 3B, 3C, and 3D alongside theother NTPase protein sequences. The identification and characterizationof the full-length CD39L2 polypeptide and nucleotide sequences isdescribed in the Example presented in Section 7, below.

To map the murine mNTPase gene, a cosmid was isolated from a mousecosmid library, and used for fluorescence in situ hybridization (FISH).For the FISH analysis, slides with mouse metaphase chromosomes wereprepared from spleens as described in Monier et al. (Monier, K. et al.,1996, Cytogenet. Cell Genet. 72:200-204). 1 microgram of mouse cosmidcontaining the mNTPase gene was labeled with biotin 14-ATP and a BionickKit (GibcoBRL). DNA was purified by passage through a Sephadex G50column and ethanol precipitated with 50 micrograms of sheared salmonsperm DNA and tRNA. 80 ng of probe was dried down with 3 micrograms ofmouse Cot-1 DNA (GibcoBRL). Hybridization was carried out as described(Ragoussis, J. et al., 1992, Genomics 14:423-430). Confirmation ofchromosomal location was achieved by rehybridizing the same slide with amouse Chromosome 12-specific Starfish Paint (Cambio).

The FISH study revealed the presence of mNTPase on mouse Chr. 12 atchromosome band E. To confirm the location of the mNTPase gene on mouseChr. 12, linkage analysis was carried out upon the EuropeanCollaborative Interspecific Backcross (EUCIB). PCR primers were designedto the 3 untranslated region of the mNTPase cDNA sequence and used forPCR by use of mouse genomic DNA from the two parental mouse strains, Musspretus and C57BL/6. A polymorphism was detected between the two strainsby SSCP analysis and was used for the mapping. (PCR conditions: 48° C.20 sec., Primer 1: CCAGACTGTAAATCTTTTGG (SEO ID NO: 20); Primer 2:AGGGAATGTAATAAGGGTAG (SEQ ID NO: 21); conditions: 94° C. 2 minutes; 35cycles of 94° C. 20 sec. 72° C. 20 sec., 72° C. 1 min; product size: 320bp).

Linkage with a LOD score of 8.14 was obtained with the genetic markerD12Mit4, flanked by D12Mit149 and D12Mit238, between 31.7 cM and 36.1 cMfrom the top of the mouse Chr. 12 linkage group for the MIT F₂Intercross. This region of mouse Chr. 12 has previously been shown toshare synteny with human Chr. 14q (DeBry, R. W. & Seldin, M. F., 1996,Genomics 33:337-351).

7. EXAMPLE Identification and Characterization of Additional NovelCD39-like Polypeptides and Nucleic Acid Molecules

Described herein is the cloning and characterization of novel CD-39-likenucleotide-triphosphatase (“NTPase”) gene and polypeptide sequences.These sequences are referred to below as “CD39L2,” “CD39L3,” “CD39L4”and “dCD39L4.”

7.1. Materials and Methods

The nucleotide sequence of CD39 (Accession No. S73813), CD39L1(Accession No. U91510), and mNTPase (see Section 6, above) were used inTBLASTX searches against entries in the expressed sequence tag (EST)database at EMBL/GenBank, using the Bork server through EMBL-Heidelberg.cDNA clones for homologous IMAGE EST entries were obtained from theHuman Genome Mapping Project Resource Centre (HGMP, Hinxton, UK). DNAwas prepared with QiaTip100 (Qiagen), and the cDNA was sequenced byprimer walking with a fluorescence labeled dye-terminator cyclesequencing kit according to the manufacturer's instructions (PRISM ReadyDye-Deoxy Terminator Premix from Applied Biosystems Inc.) andelectrophoresed on an ABI 373 (Perkin-Elmer). Overlapping EST cloneswere identified by searching with the nucleotide sequence againstentries in the EST database using BLAST-N.

Additional IMAGE cDNA clones were ordered from HGMP if they extended theexisting nucleotide sequence further 5′. cDNA clones corresponding tothe most 5′ extreme of each gene were identified by hybridization ofradiolabeled inserts of IMAGE cDNA clones to a keratinocyte stem cellcDNA library, a human adult breast epithelial cDNA library constructedusing Stratagene Lambda ZAP vector, and a Jurkat cell line cDNA libraryin pBluescript (Dunne, J. et al., 1995, Genomics 30:207-223).

Northern analysis of members of the CD39-like gene family. cDNA cloneinserts were removed by restriction digestion and separated by gelelectrophoresis. Insert DNA was gel-purified and radiolabeled (Sambrooket al., 1989, supra). Radiolabeled cDNA was prehybridized at 65° C. for2 h with 20 μg of human Cot-1 DNA (GibcoBRL) and 100 μg of total humanDNA (Sigma), before hybridization to Northern blots (Clontech, humanmultiple tissue Northern blots, Catalog No. 7760-1 and 7759-1) accordingto the manufacturer's instructions.

Members of the CD39-like gene family were mapped in the human genome byPCR screening of the GeneBridge-4 radiation hybrid mapping panelobtained from the HGMP Resource Centre (Hinxton, UK) (Gyapay, G. et al.,1996, Hum. Mol. Genet. 5:339-346). PCR-positive radiation hybrid cloneswere organized into the GeneBridge-4 HGMP-RC subset order using the HGMPradiation hybrid mapping World Wide Web (WWW) site and mapping data foreach gene were obtained from the Whitehead server. The chromosomallocation for each gene was confirmed by PCR screening of themonochromosomal hybrids obtained from the HGMP Resource Centre. PCRprimers were designed for the 3′ untranslated region (UTR) of each geneand titrated for a unique human-specific PCR product. PCR conditions:CD39L2, Primer 1, 5′-CTGCTTGAGTGACGTCTCTG-3′ (SEQ ID NO: 22); Primer 2,5′-CACATGAGGTTCAGCTCGTG-3′ (SEQ ID NO: 23); 94° C. for 2 min; 38 cyclesof 94° C. for 20 s, 54° C. for 20 s, 72° C. for 20 s; 72° C. for 2 min.Product size is 362 bp). CD39L3, Primer 1: 5′-GTGAAGTGGCTGCCTTCAGG-3′(SEQ ID NO: 24); Primer 2, 5′-CCTTTGACTCGGGACTCCAG-3′ (SEQ ID NO: 25);94° C. for 2 min; 38 cycles of 94° C. for 20 s, 56° C. for 20 s, 72° C.for 2 min. Product size is 281 bp). CD39L4. Primer 1,5′-GAACTGCTGCCTAACCACTC-3′ (SEQ ID NO: 26); Primer 2,5′-ATTGATGGGTCTTGGGATTGC-3′ (SEQ ID NO: 27); 94° C. 2 for min; 38 cyclesof 94° C. for 20 s, 56° C. for 20 s; 72° C. for 20 s; 72° C. for 2 min.Product size is 234 bp. PCR products were analyzed by electrophoresisthrough 3.5% NuSieve agarose gels (Flowgen).

7.2. Results

Isolation and Sequence Characterization of CD39L2.

Identification of partial human CD39L2 sequence was described in theExample presented in Section 6, above. The CD39L2 insert was used toisolate additional clones from a human adult breast epithelial cDNAlibrary (ZR75), a human T-leukemia cell line J6 cDNA library (Jurkat),and a human keratinocyte stem cell cDNA library (KER). Of 23 cDNA clonesthat were isolated and sequenced, all but one appeared to bealternatively spliced or unspliced. Within the 2762 bp cDNA thatappeared to be neither unspliced or alternatively spliced, an ORFextending to nucleotide 1600 containing ACRs I-IV was identified. TwoATG codons with a poor match to the consensus translation initiationsite were found at nucleotide positions 148 and 232 (AUGUGAAUGA (SEQ IDNO: 28) at 148 and ACAAGGAUGA (SEQ ID NO: 29) at 232 versus consensusGCCGCCAUGG (SEQ ID NO: 19); Kozak, M., 1989, J. Biol. Chem.108:229-241). Based on homology to mNTPase, the ATG at nucleotideposition 232 is the initiation codon. (See FIGS. 9A, 9B, 9C, 9D, and 9Efor a depiction of the CD39L2 amino acid sequence that results fromtranslation from the upstream, position 148, start codon; such a form ofCD39L2 as well as nucleotide sequences that encode this form of thepolypeptide are also intended to be included as part of the presentinvention.) A single polyadenylation signal of AAUAAA (SEQ ID NO: 30)was identified at nucleotide position 2700, 22 nucleotides 5′ of thepoly(A) tail of the human CD39L2 cDNA.

The nucleotide sequence (SEQ ID NO:1) and derived amino acid sequence(SEQ ID NO:2) of human CD39L2 is depicted in FIGS. 4A, 4B, 4C, 4D, 4E,4F, 4G, and 4H. Hydrophobicity plots using Topred-II 1.1 (Claros, M. G.& Von Hejine, G., 1994, Comput. Appl. Biosci. 10:685-686) predicted asingle transmembrane segment at the N-terminal extreme of the protein,suggesting that CD39L2 has a short putative cytoplasmic tail and a largeextracellular C-terminal domain (FIG. 5C). There are two potentialN-glycosylation sites in the predicted extracellular domain. A cAMP andcGMP-dependent protein kinase and a protein kinase-C phosphorylationsite are found directly after the initiation methionine codon(nucleotide 232).

Isolation and Sequence Characterization of CD39L3. An additional novelgene and polypeptide CD39-like sequence, referred to herein as CD39L3,was also identified. BLASTN search of the NCBI EST database with thefull cDNA sequence for human CD39 (Accession No. S73813) identifiedthree EST entries for cDNA clones from an endometrial tumor library(Accession Nos. AA336644, AA338117, and AA337885), which led to theidentification of an IMAGE library EST (Accession No. N72742) that wascompletely sequenced and had amino acid homology to CD39 and CD39L1. Theinsert of N72742 was used to screen the Jurkat, ZR75, and KER cDNAlibraries, and a single cDNA clone was isolated from the KER library andsequenced.

A 1669-bp ORF was identified within the cDNA insert. The nucleotidesequence (SEQ ID NO:3) and derived amino acid sequence (SEQ ID NO:4) ofthe cDNA insert, referred to herein as CD39L3, are shown in FIGS. 6A,6B, 6C, 6D, 6E, 6F, 6G, and 6H. The amino acid sequence was revealed tocontain ACRs I-IV, an ATG codon at position 83, and a singlepolyadenylation signal at position 2758. Hydrophobicity plots asdescribed above predict two potential transmembrane segments at the Nand C-terminal extremes of the protein (FIG. 5D). There are sevenpotential extracellular N-glycosylation sites. A cAMP- andcGMP-dependent protein kinase site and a protein kinase-Cphosphorylation site are located at the C-terminal extreme of theprotein.

An additional (CD39-like gene and polypeptile sequence, referred toherein as CD39L4, was also identified. A TBLASTX search of the NCBI ESTdatabase with the full cDNA sequence for the mNTPase was performed. Ahuman EST clone was sequenced, and an ORF was identified extending tonucleotide 529 of 2260 nucleotides that contained ACR I only and an ATGcodon at position 256. In the same reading frame, downstream of the stopcodon at nucleotide 529, an ORF extending to nucleotide 1792 containedACRs II, III, and IV. Further analysis of the nucleotide sequencerevealed a putative intron with splice donor and acceptor sites thatconform to the 5′ gt . . . 3′ ag rule (Breathnach and Chambon, 1981, AnnRev. Biochem. 50:349-383; splice donor CAGgtcacttatggagcctg (SEQ ID NO:31) at nucleotide position 470, acceptor ccatggacaaaatagGAC (SEQ ID NO:32) at position 710, exon sequence underlined). Further analysis of thesequence revealed that removal of the 251-bp putative intron wouldresult in a contiguous ORF containing ACRs I-IV. The hypothesis thatthis sequence does indeed constitute an intron was only confirmed byisolation and sequencing of three additional cDNA clones (CD39L4J1-3)from the Jurkat library, one of which contained the 251 bp.

The nucleotide sequence (SEQ ID NO:5) and derived amino acid sequence(SEQ ID NO:6), referred to herein as CD39L4, is depicted in FIGS. 7A,7B, 7C, 7D, 7E, and 7F. The sequence contained a poly(A)tail, but noconsensus polyadenylation sequence (Proudfoot, 1991, New Biol.3:851-854). This is also the case for mNTPase. Hydrophobicity plots asdescribed above predicted a single transmembrane segment at theN-terminal extreme of the protein (FIG. 5E). This is similar to thepredicted topology of CD39L2 and different from that of CD39, Cd39L1,and CD39L3. There are three potential extracellular N-glycosylationsites.

FIGS. 8A, 8B, 8C, and 8D depicts an alignment of each of theabove-described sequences, along with other members of the NTPaseCD-39-like gene family. The ACR domains are indicated by arrows.

Expression of the Human Members of the CD39-like Family.

Representative probes for each member of the CD39-like gene family werehybridized to human multiple tissue Northern blots. Results arepresented below.

CD39L2. Hybridization of the CD39L2 cDNA insert to the multiple tissueNorthern blots resulted in two prominent signals of 2.6 kb (major) and4.4 kb (minor) in all tissues studied. This is most likely due todifferential polyadenylation.

CD39L3. A PCR product covering the coding sequence of CD39L3 was usedfor the Northern hybridizations. A strong signal of approximately 3.0 kbcould be seen in adult brain, pancreas, spleen, and prostate. Thoughmoderate or low expression was seen in most other tissues, no signal wasdetected in adult liver and peripheral blood leukocytes. A weaker signalof approximately 1.8 kb was found in adult pancreas and may be theresult of alternative splicing.

CD39L4. The CD39L4 cDNA was hybridized to the same Northern blots, and aprominent signal of approximately 4.8 kb was seen in adult liver,kidney, prostate, testis, and colon. Considerably weaker expression wasseen for all other tissues examined. Several smaller bands were observedin tissues showing the strongest expression of CD39L4 and may be theresult of differential polyadenylation or alternative splicing.

Mapping of Members of the CD39-like Family.

The CD39L2 gene was mapped with a lod score of >19 to human chromosome20 by PCR typing of the GeneBridge 4 Radiation Hybrid Mapping Panel(Gyapay et al., 1996, Human Mol. Genet. 5:339-346). CD39L2 mapped 9.76cR from D20S493 (typing data: 12012 02101 22000 00111 00110 01210 0011001101 10121 00100 00120 11211 00011 11012 01001 01102 00000 00000 001).Using the closet flanking markers (D20S184 and D20S99) also representedon the consensus map, this placed CD39L2 at chromosome band 20q11.2. Thelocation of CD39L2 on human chromosome 20 was confirmed by PCR analysisof monochromosomal mapping panels (Kelsell et al., 1995, Ann. HumanGenet. 59:233-241). On the basis of synteny to human chromosome 20q11.2,the mouse homolog of CD39L2 was expected to map to mouse chromosome 2(DeBry and Seldin, 1996, Genomics 3:337-351).

The CD39L3 gene was mapped as described above to human chromosome 3,5.76 cR from D3S3390 (data: 12002 02010 22000 00011 20000 00110 0100100000 02022 11000 10001 00200 21100 00212 01010 10002 00000 00011 001).Using the closest flanking markers as described above (D3S1561 andD3S3564), this placed CD39L3 at chromosome band 3p21.3. The location ofCD39L3 on chromosome 3 was confirmed by PCR as for CD39L2. On the basisof synteny, the mouse homologue of Cd39L3 was expected to map to mousechromosome 9 (DeBry and Seldin, 1996, Genomics 33:337-351).

The CD39L4 gene was mapped as described above to human chromosome 14,1.92 cR from D14S71 (data: 02102 02102 22000 01010 11021 01000 0101010110 02121 21000 00010 00211 01001 10102 02012 00002 12111 01100 002).This placed CD39L4 at chromosome band 14q24. The chromosomal location ofCD39L4 was confirmed as described above.

Identification of a Drosophila Gene with high Nomolocv to CD39L2 andCD39L4. A D. melanogaster CD39-like gene was also identified. A TBLASTXsearch of the EST database using the human CD39L2 cDNA sequence, fiveDrosophila EST entries were identified (Accession No. AA391695,AA390461, AA201196, AA246996, and AA567512). A consensus sequence wasgenerated and used for a BLASTN search against EMBL/GenBank entries. Asingle D. melanogaster genomic entry (Accession No. AC002032) wasidentified showing 100% sequence identity to three regions of the ESTconsensus sequence. Alignment of the EST consensus against the genomicsequence identified three exons that conform to the 5′ gt . . . 3′ agrule (Breathnach & Chambon, 1981, Ann. Rev. Biochem. 50:349-383). Exon 4was identified on the basis of reading frame homology to the CD39L2 andCD39L4 proteins. An ATG codon was identified in exon 1, a stop codon inexon 4.

The predicted amino acid sequence of the D. melanogaster CD39-like gene,referred to herein as dCD39L4, containing the ACRs-I-IV was shown inFIGS. 9A, 9B, 9C, 9D, and 9E, aligned against the gene family memberswith the highest homology. Three N-glycosylation consensus sites werefound in the putative extracelluar domain, and two potential cAMP- andc-GMP-dependent protein kinase phosphorylation sites were found in theputative N-terminal cytoplasmic domain. Hydrophobicity plots asdescribed above predicted a single transmembrane segment at theN-terminal extreme of the dCD39L4 protein. The topology of dCD39L4 istherefore most similar to the predicted topology of the CD39L2 andCd39L4 proteins.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention, and compositions and methods which are functionallyequivalent are within the scope of the invention. Indeed, numerousmodifications and variations in the practice of the invention areexpected to occur to those skilled in the art upon consideration of thepresent preferred embodiments. Consequently, the only limitations whichshould be placed upon the scope of the invention are those which appearin the appended claims.

All references cited within the body of the instant specification arehereby incorporated by reference in their entirety.

32 1 2762 DNA Homo Sapiens CDS (232)..(1599) 1 gtggggtcgt atcccgcgggtggaggccgg ggtggcgccg gccggggcgg gggagcccaa 60 aagaccggct gccgcctgctccccggaaaa gggcactcgt ctccgtgggt gtggcggagc 120 gcgcggtgca tggaatgggctatgtgaatg aaaaaaggta tccgttatga aacttccaga 180 aaaacgagct acatttttcagcagccgcag cacggtcctt ggcaaacaag g atg aga 237 Met Arg 1 aaa ata tcc aaccac ggg agc ctg cgg gtg gcg aag gtg gca tac ccc 285 Lys Ile Ser Asn HisGly Ser Leu Arg Val Ala Lys Val Ala Tyr Pro 5 10 15 ctg ggg ctg tgt gtgggc gtg ttc atc tat gtt gcc tac atc aag tgg 333 Leu Gly Leu Cys Val GlyVal Phe Ile Tyr Val Ala Tyr Ile Lys Trp 20 25 30 cac cgg gcc acc gcc acccag gcc ttc ttc agc atc acc agg gca gcc 381 His Arg Ala Thr Ala Thr GlnAla Phe Phe Ser Ile Thr Arg Ala Ala 35 40 45 50 ccg ggg gcc cgg tgg ggtcag cag gcc cac agc ccc ctg ggg aca gct 429 Pro Gly Ala Arg Trp Gly GlnGln Ala His Ser Pro Leu Gly Thr Ala 55 60 65 gca gac ggg cac gag gtc ttctac ggg atc atg ttt gat gca gga agc 477 Ala Asp Gly His Glu Val Phe TyrGly Ile Met Phe Asp Ala Gly Ser 70 75 80 act ggc acc cga gta cac gtc ttccag ttc acc cgg ccc ccc aga gaa 525 Thr Gly Thr Arg Val His Val Phe GlnPhe Thr Arg Pro Pro Arg Glu 85 90 95 act ccc acg tta acc cac gaa acc ttcaaa gca gtg aag cca ggt ctt 573 Thr Pro Thr Leu Thr His Glu Thr Phe LysAla Val Lys Pro Gly Leu 100 105 110 tct gcc tat gct gat gat gtt gaa aagagc gct cag gga atc cgg gaa 621 Ser Ala Tyr Ala Asp Asp Val Glu Lys SerAla Gln Gly Ile Arg Glu 115 120 125 130 cta ctg gat gtt gct aaa cag gacatt ccg ttc gac ttc tgg aag gcc 669 Leu Leu Asp Val Ala Lys Gln Asp IlePro Phe Asp Phe Trp Lys Ala 135 140 145 acc cct ctg gtc ctc aag gcc acagct ggc tta cgc ctg tta cct gga 717 Thr Pro Leu Val Leu Lys Ala Thr AlaGly Leu Arg Leu Leu Pro Gly 150 155 160 gaa aag gcc cag aag tta ctg cagaag gtg aaa gaa gta ttt aaa gca 765 Glu Lys Ala Gln Lys Leu Leu Gln LysVal Lys Glu Val Phe Lys Ala 165 170 175 tcg cct ttc ctt gta ggg gat gactgt gtt tcc atc atg aac gga aca 813 Ser Pro Phe Leu Val Gly Asp Asp CysVal Ser Ile Met Asn Gly Thr 180 185 190 gat gaa ggc gtt tcg gcg tgg atcacc atc aac ttc ctg aca ggc agc 861 Asp Glu Gly Val Ser Ala Trp Ile ThrIle Asn Phe Leu Thr Gly Ser 195 200 205 210 ttg aaa act cca gga ggg agcagc gtg ggc atg ctg gac ttg ggc gga 909 Leu Lys Thr Pro Gly Gly Ser SerVal Gly Met Leu Asp Leu Gly Gly 215 220 225 gga tcc act cag atc gcc ttcctg cca cgc gtg gag ggc acc ctg cag 957 Gly Ser Thr Gln Ile Ala Phe LeuPro Arg Val Glu Gly Thr Leu Gln 230 235 240 gcc tcc cca ccc ggc tac ctgacg gca ctg cgg atg ttt aac agg acc 1005 Ala Ser Pro Pro Gly Tyr Leu ThrAla Leu Arg Met Phe Asn Arg Thr 245 250 255 tac aag ctc tat tcc tac agctac ctc ggg ctc ggg ctg atg tcg gca 1053 Tyr Lys Leu Tyr Ser Tyr Ser TyrLeu Gly Leu Gly Leu Met Ser Ala 260 265 270 cgc ctg gcg atc ctg ggc ggcgtg gag ggg cag cct gct aag gat gga 1101 Arg Leu Ala Ile Leu Gly Gly ValGlu Gly Gln Pro Ala Lys Asp Gly 275 280 285 290 aag gag ttg gtc agc ccttgc ttg tct ccc agt ttc aaa gga gag tgg 1149 Lys Glu Leu Val Ser Pro CysLeu Ser Pro Ser Phe Lys Gly Glu Trp 295 300 305 gaa cac gca gaa gtc acgtac agg gtt tca ggg cag aaa gca gcg gca 1197 Glu His Ala Glu Val Thr TyrArg Val Ser Gly Gln Lys Ala Ala Ala 310 315 320 agc ctg cac gag ctg tgtgct gcc aga gtg tca gag gtc ctt caa aac 1245 Ser Leu His Glu Leu Cys AlaAla Arg Val Ser Glu Val Leu Gln Asn 325 330 335 aga gtg cac agg acg gaggaa gtg aag cat gtg gac ttc tat gct ttc 1293 Arg Val His Arg Thr Glu GluVal Lys His Val Asp Phe Tyr Ala Phe 340 345 350 tcc tac tat tac gac cttgca gct ggt gtg ggc ctc ata gat gcg gag 1341 Ser Tyr Tyr Tyr Asp Leu AlaAla Gly Val Gly Leu Ile Asp Ala Glu 355 360 365 370 aag gga ggc agc ctggtg gtg ggg gac ttc gag atc gca gcc aag tac 1389 Lys Gly Gly Ser Leu ValVal Gly Asp Phe Glu Ile Ala Ala Lys Tyr 375 380 385 gtg tgt cgg acc ctggag aca cag ccg cag agc agc ccc ttc tca tgc 1437 Val Cys Arg Thr Leu GluThr Gln Pro Gln Ser Ser Pro Phe Ser Cys 390 395 400 atg gac ctc acc tacgtc agc ctg cta ctc cag gag ttc ggc ttt ccc 1485 Met Asp Leu Thr Tyr ValSer Leu Leu Leu Gln Glu Phe Gly Phe Pro 405 410 415 agg agc aaa gtg ctgaag ctc act cgg aaa att gac aat gtt gag acc 1533 Arg Ser Lys Val Leu LysLeu Thr Arg Lys Ile Asp Asn Val Glu Thr 420 425 430 agc tgg gct ctg ggggcc att ttt cat tac atc gac tcc ctg aac aga 1581 Ser Trp Ala Leu Gly AlaIle Phe His Tyr Ile Asp Ser Leu Asn Arg 435 440 445 450 cag aag agt ccagcc tca tagtggccga gccatccctg tccccgtcag 1629 Gln Lys Ser Pro Ala Ser455 cagtgtctgt gtgtctgcat aaaccctcct gtcctggacg tgacttcatc ctgaggagcc1689 acagcacagg ccgtgctggc actttctgca cactggctct gggacttgca gaaggcctgg1749 tgctgccctg gcatcagcct cttccagtca catctggcca gagggctgtc tggacctggg1809 ccctgctcaa tgccacctgt ctgcctgggc tccaagtggg caggaccagg acagaaccac1869 aggcacacac tgagggggca gtgtggctcc ctgcctgtcc catccccatg ccccgtccgc1929 ggggctgtgg ctgctgctgt gcatgtccct gcgatgggag tcttgtctcc cagcctgtca1989 gtttcctccc cagggcagag ctccccttcc tgcaagagtc tgggaggcgg tgcaggctgt2049 cctggctgct ctggggaagc cgagggacag ccataacacc cccgggacag taggtctggg2109 cggcaccact gggaactctg gacttgagtg tgtttgctct tccttgggta tgaatgtgtg2169 agttcaccca gaggcctgct ctcctcacac attgtgtggt ttggggttaa tgatggaggg2229 agacacctct tcatagacgg caggtgccca cctttcaggg agtctcccag catgggcgga2289 tgccgggcat gagctgctgt aaactatttg tggctgtgct gcttgagtga cgtctctgtc2349 gtgtgggtgc caagtgcttg tgtagaaact gtgttctgag cccccttttc tggacaccaa2409 ctgtgtcctg tgaatgtatc gctactgtga gctgttcccg cctagccagg gccatgtctt2469 aggtgcagct gtgccacggg tcagctgagc cacagtccca gaaccaagct ctcggtgtct2529 cgggccacca tccgcccacc tcgggctgac cccacctcct ccatggacag tgtgagcccc2589 gggccgtgca tcctgctcag tgtggcgtca gtgtcggggc tgagcccctt gagctgcttc2649 agtgaatgta cagtgcccgg cacgagctga acctcatgtg ttccactccc aataaaaggt2709 tgacaggggc ttctccttca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 2762 2456 PRT Homo Sapiens 2 Met Arg Lys Ile Ser Asn His Gly Ser Leu Arg ValAla Lys Val Ala 1 5 10 15 Tyr Pro Leu Gly Leu Cys Val Gly Val Phe IleTyr Val Ala Tyr Ile 20 25 30 Lys Trp His Arg Ala Thr Ala Thr Gln Ala PhePhe Ser Ile Thr Arg 35 40 45 Ala Ala Pro Gly Ala Arg Trp Gly Gln Gln AlaHis Ser Pro Leu Gly 50 55 60 Thr Ala Ala Asp Gly His Glu Val Phe Tyr GlyIle Met Phe Asp Ala 65 70 75 80 Gly Ser Thr Gly Thr Arg Val His Val PheGln Phe Thr Arg Pro Pro 85 90 95 Arg Glu Thr Pro Thr Leu Thr His Glu ThrPhe Lys Ala Val Lys Pro 100 105 110 Gly Leu Ser Ala Tyr Ala Asp Asp ValGlu Lys Ser Ala Gln Gly Ile 115 120 125 Arg Glu Leu Leu Asp Val Ala LysGln Asp Ile Pro Phe Asp Phe Trp 130 135 140 Lys Ala Thr Pro Leu Val LeuLys Ala Thr Ala Gly Leu Arg Leu Leu 145 150 155 160 Pro Gly Glu Lys AlaGln Lys Leu Leu Gln Lys Val Lys Glu Val Phe 165 170 175 Lys Ala Ser ProPhe Leu Val Gly Asp Asp Cys Val Ser Ile Met Asn 180 185 190 Gly Thr AspGlu Gly Val Ser Ala Trp Ile Thr Ile Asn Phe Leu Thr 195 200 205 Gly SerLeu Lys Thr Pro Gly Gly Ser Ser Val Gly Met Leu Asp Leu 210 215 220 GlyGly Gly Ser Thr Gln Ile Ala Phe Leu Pro Arg Val Glu Gly Thr 225 230 235240 Leu Gln Ala Ser Pro Pro Gly Tyr Leu Thr Ala Leu Arg Met Phe Asn 245250 255 Arg Thr Tyr Lys Leu Tyr Ser Tyr Ser Tyr Leu Gly Leu Gly Leu Met260 265 270 Ser Ala Arg Leu Ala Ile Leu Gly Gly Val Glu Gly Gln Pro AlaLys 275 280 285 Asp Gly Lys Glu Leu Val Ser Pro Cys Leu Ser Pro Ser PheLys Gly 290 295 300 Glu Trp Glu His Ala Glu Val Thr Tyr Arg Val Ser GlyGln Lys Ala 305 310 315 320 Ala Ala Ser Leu His Glu Leu Cys Ala Ala ArgVal Ser Glu Val Leu 325 330 335 Gln Asn Arg Val His Arg Thr Glu Glu ValLys His Val Asp Phe Tyr 340 345 350 Ala Phe Ser Tyr Tyr Tyr Asp Leu AlaAla Gly Val Gly Leu Ile Asp 355 360 365 Ala Glu Lys Gly Gly Ser Leu ValVal Gly Asp Phe Glu Ile Ala Ala 370 375 380 Lys Tyr Val Cys Arg Thr LeuGlu Thr Gln Pro Gln Ser Ser Pro Phe 385 390 395 400 Ser Cys Met Asp LeuThr Tyr Val Ser Leu Leu Leu Gln Glu Phe Gly 405 410 415 Phe Pro Arg SerLys Val Leu Lys Leu Thr Arg Lys Ile Asp Asn Val 420 425 430 Glu Thr SerTrp Ala Leu Gly Ala Ile Phe His Tyr Ile Asp Ser Leu 435 440 445 Asn ArgGln Lys Ser Pro Ala Ser 450 455 3 2797 DNA Homo Sapiens CDS (83)..(1669)3 acccacgcgt ctggccgcgg gccgcctctg cggcagcgct agtcgccttc tccgaatcgg 60ctccgcacag ctaggagaaa ag atg ttc act gtg ctg acc cgc caa cca tgt 112 MetPhe Thr Val Leu Thr Arg Gln Pro Cys 1 5 10 gag caa gca ggc ctc aag gccctc tac cga act cca acc atc att gcc 160 Glu Gln Ala Gly Leu Lys Ala LeuTyr Arg Thr Pro Thr Ile Ile Ala 15 20 25 ttg gtg gtc ttg ctt gtg agt attgtg gta ctt gtg agt atc act gtc 208 Leu Val Val Leu Leu Val Ser Ile ValVal Leu Val Ser Ile Thr Val 30 35 40 atc cag atc cac aag caa gag gtc ctccct cca gga ctg aag tat ggt 256 Ile Gln Ile His Lys Gln Glu Val Leu ProPro Gly Leu Lys Tyr Gly 45 50 55 att gtg ctg gat gcc ggg tct tca aga accaca gtc tac gtg tat caa 304 Ile Val Leu Asp Ala Gly Ser Ser Arg Thr ThrVal Tyr Val Tyr Gln 60 65 70 tgg cca gca gaa aaa gag aat aat acc gga gtggtc agt caa acc ttc 352 Trp Pro Ala Glu Lys Glu Asn Asn Thr Gly Val ValSer Gln Thr Phe 75 80 85 90 aaa tgt agt gtg aaa ggc tct gga atc tcc agctat gga aat aac ccc 400 Lys Cys Ser Val Lys Gly Ser Gly Ile Ser Ser TyrGly Asn Asn Pro 95 100 105 caa gat gtc ccc aga gcc ttt gag gag tgt atgcaa aaa gtc aag ggg 448 Gln Asp Val Pro Arg Ala Phe Glu Glu Cys Met GlnLys Val Lys Gly 110 115 120 cag gtt cca tcc cac ctc cac gga tcc acc cccatt cac ctg gga gcc 496 Gln Val Pro Ser His Leu His Gly Ser Thr Pro IleHis Leu Gly Ala 125 130 135 acg gct ggg atg cgc ttg ctg agg ttg caa aatgaa aca gca gct aat 544 Thr Ala Gly Met Arg Leu Leu Arg Leu Gln Asn GluThr Ala Ala Asn 140 145 150 gaa gtc ctt gaa agc atc caa agc tac ttc aagtcc cag ccc ttt gac 592 Glu Val Leu Glu Ser Ile Gln Ser Tyr Phe Lys SerGln Pro Phe Asp 155 160 165 170 ttt agg ggt gct caa atc att tct ggg caagaa gaa ggg gta tat gga 640 Phe Arg Gly Ala Gln Ile Ile Ser Gly Gln GluGlu Gly Val Tyr Gly 175 180 185 tgg att aca gcc aac tat tta atg gga aatttc ctg gag aag aac ctg 688 Trp Ile Thr Ala Asn Tyr Leu Met Gly Asn PheLeu Glu Lys Asn Leu 190 195 200 tgg cac atg tgg gtg cac ccg cat gga gtggaa acc acg ggt gcc ctg 736 Trp His Met Trp Val His Pro His Gly Val GluThr Thr Gly Ala Leu 205 210 215 gac tta ggt ggt gcc tcc acc caa ata tccttc gtg gca gga gag aag 784 Asp Leu Gly Gly Ala Ser Thr Gln Ile Ser PheVal Ala Gly Glu Lys 220 225 230 atg gat ctg aac acc agc gac atc atg caggtg tcc ctg tat ggc tac 832 Met Asp Leu Asn Thr Ser Asp Ile Met Gln ValSer Leu Tyr Gly Tyr 235 240 245 250 gta tac acg ctc tac aca cac agc ttccag tgc tat ggc cgg aat gag 880 Val Tyr Thr Leu Tyr Thr His Ser Phe GlnCys Tyr Gly Arg Asn Glu 255 260 265 gct gag aag aag ttt ctg gca atg ctcctg cag aat tct cct acc aaa 928 Ala Glu Lys Lys Phe Leu Ala Met Leu LeuGln Asn Ser Pro Thr Lys 270 275 280 aac cat ctc acc aat ccc tgt tac cctcgg gat tat agc atc agc ttc 976 Asn His Leu Thr Asn Pro Cys Tyr Pro ArgAsp Tyr Ser Ile Ser Phe 285 290 295 acc atg ggc cat gta ttt gat agc ctgtgc act gtg gac cag agg cca 1024 Thr Met Gly His Val Phe Asp Ser Leu CysThr Val Asp Gln Arg Pro 300 305 310 gaa agt tat aac ccc aat gat gtc atcact ttt gaa gga act ggg gac 1072 Glu Ser Tyr Asn Pro Asn Asp Val Ile ThrPhe Glu Gly Thr Gly Asp 315 320 325 330 cca tct ctg tgt aag gag aag gtggct tcc ata ttt gac ttc aaa gct 1120 Pro Ser Leu Cys Lys Glu Lys Val AlaSer Ile Phe Asp Phe Lys Ala 335 340 345 tgc cat gat caa gaa acc tgt tctttt gat ggg gtt tat cag cca aag 1168 Cys His Asp Gln Glu Thr Cys Ser PheAsp Gly Val Tyr Gln Pro Lys 350 355 360 att aaa ggg cca ttt gtg gct tttgca gga ttc tac tac aca gcc agt 1216 Ile Lys Gly Pro Phe Val Ala Phe AlaGly Phe Tyr Tyr Thr Ala Ser 365 370 375 gct tta aat ctt tca ggt agc ttttcc ctg gac acc ttc aac tcc agc 1264 Ala Leu Asn Leu Ser Gly Ser Phe SerLeu Asp Thr Phe Asn Ser Ser 380 385 390 acc tgg aat ttc tgc tca cag aattgg agt cag ctc cca ctg ctg ctc 1312 Thr Trp Asn Phe Cys Ser Gln Asn TrpSer Gln Leu Pro Leu Leu Leu 395 400 405 410 ccc aaa ttt gat gag gta tatgcc cgc tct tac tgc ttc tca gcc aac 1360 Pro Lys Phe Asp Glu Val Tyr AlaArg Ser Tyr Cys Phe Ser Ala Asn 415 420 425 tac atc tac cac ttg ttt gtgaac ggt tac aaa ttc aca gag gag act 1408 Tyr Ile Tyr His Leu Phe Val AsnGly Tyr Lys Phe Thr Glu Glu Thr 430 435 440 tgg ccc caa ata cac ttt gaaaaa gaa gtg ggg aat agc agc ata gcc 1456 Trp Pro Gln Ile His Phe Glu LysGlu Val Gly Asn Ser Ser Ile Ala 445 450 455 tgg tct ctt ggc tac atg ctcagc ctg acc aac cag atc cca gct gaa 1504 Trp Ser Leu Gly Tyr Met Leu SerLeu Thr Asn Gln Ile Pro Ala Glu 460 465 470 agc cct ctg atc cgt ctg cccata gaa cca cct gtc ttt gtg ggc acc 1552 Ser Pro Leu Ile Arg Leu Pro IleGlu Pro Pro Val Phe Val Gly Thr 475 480 485 490 ctc gct ttc ttc aca gtggca gcc ttg ctg tgt ctg gca ttt ctt gca 1600 Leu Ala Phe Phe Thr Val AlaAla Leu Leu Cys Leu Ala Phe Leu Ala 495 500 505 tac ctg tgt tca gca accaga aga aag agg cac tcc gag cat gcc ttt 1648 Tyr Leu Cys Ser Ala Thr ArgArg Lys Arg His Ser Glu His Ala Phe 510 515 520 gac cat gca gtg gat tctgac tgagccttca aagcagctcc tggagtccaa 1699 Asp His Ala Val Asp Ser Asp525 tggctgctta gagtcagcct gggtggcacc aggcaatgca ggtgaagtgg ctgccttcag1759 gaaatacaac taactaaaat caaacaccta ggtcacgtgc ctctcaaata ctgatttctg1819 ccacagcacc tcttgaggca tcccttggct attctgtgca tattgttctt cagagacctc1879 actacccaca tgctgatcta ttggggaaca gagaagagac aggccactaa ggtcaggctc1939 tttatattaa gttccccaga ggaagagtaa gttgagaagg tatcagttta atgttgaaga1999 attgacctca gggctcagtt tccatttccc tccctcagta ttcttcctgg caagataccc2059 attaagcatt tcgccaatca gaatctcatt ttatagtttt tcccattggt ctttaactaa2119 gactttcttg tagcaatctc gtaagcagtg aaccccctca gatcagtaga atatagtatc2179 tgggggagaa gacttacttc cttcagggca gcagccacag ccaggcttct gtcatacagg2239 tagatcccga agcacagaga cataaaaaag gtctcccaga aaactataga ccattctcca2299 agtggaattc ccacttaggg ctctggtcac tagattgcaa cctgtgtgtt tgtcatcatc2359 ctcatctcac cattgtattg ctatgccctc ccataaaaac acattgatcc ctagcaagat2419 tattgcattc cagattttac tgcctttgct aggcttttgc ttagcaaagg gctgactttc2479 cattgttatc atggtgtata tatttttgtc accattccca caagtatact tgatgttgtc2539 atagaacgaa catcctactc tatgatttac taaccaatta ctttcccaga tcatagacct2599 ctctgcatag tagtcatagg tcttgacttt ggggaaagaa aaggaagctg caggaatatt2659 tatctccaaa gtcgaatgag aaagaactcc agcaaatcca atggctacaa actaaaaatc2719 agcattattt catattgctg tttcttagct gaatatggaa taaagaacta ttattttatt2779 ttgaaaaaaa aaaaaaaa 2797 4 529 PRT Homo Sapiens 4 Met Phe Thr ValLeu Thr Arg Gln Pro Cys Glu Gln Ala Gly Leu Lys 1 5 10 15 Ala Leu TyrArg Thr Pro Thr Ile Ile Ala Leu Val Val Leu Leu Val 20 25 30 Ser Ile ValVal Leu Val Ser Ile Thr Val Ile Gln Ile His Lys Gln 35 40 45 Glu Val LeuPro Pro Gly Leu Lys Tyr Gly Ile Val Leu Asp Ala Gly 50 55 60 Ser Ser ArgThr Thr Val Tyr Val Tyr Gln Trp Pro Ala Glu Lys Glu 65 70 75 80 Asn AsnThr Gly Val Val Ser Gln Thr Phe Lys Cys Ser Val Lys Gly 85 90 95 Ser GlyIle Ser Ser Tyr Gly Asn Asn Pro Gln Asp Val Pro Arg Ala 100 105 110 PheGlu Glu Cys Met Gln Lys Val Lys Gly Gln Val Pro Ser His Leu 115 120 125His Gly Ser Thr Pro Ile His Leu Gly Ala Thr Ala Gly Met Arg Leu 130 135140 Leu Arg Leu Gln Asn Glu Thr Ala Ala Asn Glu Val Leu Glu Ser Ile 145150 155 160 Gln Ser Tyr Phe Lys Ser Gln Pro Phe Asp Phe Arg Gly Ala GlnIle 165 170 175 Ile Ser Gly Gln Glu Glu Gly Val Tyr Gly Trp Ile Thr AlaAsn Tyr 180 185 190 Leu Met Gly Asn Phe Leu Glu Lys Asn Leu Trp His MetTrp Val His 195 200 205 Pro His Gly Val Glu Thr Thr Gly Ala Leu Asp LeuGly Gly Ala Ser 210 215 220 Thr Gln Ile Ser Phe Val Ala Gly Glu Lys MetAsp Leu Asn Thr Ser 225 230 235 240 Asp Ile Met Gln Val Ser Leu Tyr GlyTyr Val Tyr Thr Leu Tyr Thr 245 250 255 His Ser Phe Gln Cys Tyr Gly ArgAsn Glu Ala Glu Lys Lys Phe Leu 260 265 270 Ala Met Leu Leu Gln Asn SerPro Thr Lys Asn His Leu Thr Asn Pro 275 280 285 Cys Tyr Pro Arg Asp TyrSer Ile Ser Phe Thr Met Gly His Val Phe 290 295 300 Asp Ser Leu Cys ThrVal Asp Gln Arg Pro Glu Ser Tyr Asn Pro Asn 305 310 315 320 Asp Val IleThr Phe Glu Gly Thr Gly Asp Pro Ser Leu Cys Lys Glu 325 330 335 Lys ValAla Ser Ile Phe Asp Phe Lys Ala Cys His Asp Gln Glu Thr 340 345 350 CysSer Phe Asp Gly Val Tyr Gln Pro Lys Ile Lys Gly Pro Phe Val 355 360 365Ala Phe Ala Gly Phe Tyr Tyr Thr Ala Ser Ala Leu Asn Leu Ser Gly 370 375380 Ser Phe Ser Leu Asp Thr Phe Asn Ser Ser Thr Trp Asn Phe Cys Ser 385390 395 400 Gln Asn Trp Ser Gln Leu Pro Leu Leu Leu Pro Lys Phe Asp GluVal 405 410 415 Tyr Ala Arg Ser Tyr Cys Phe Ser Ala Asn Tyr Ile Tyr HisLeu Phe 420 425 430 Val Asn Gly Tyr Lys Phe Thr Glu Glu Thr Trp Pro GlnIle His Phe 435 440 445 Glu Lys Glu Val Gly Asn Ser Ser Ile Ala Trp SerLeu Gly Tyr Met 450 455 460 Leu Ser Leu Thr Asn Gln Ile Pro Ala Glu SerPro Leu Ile Arg Leu 465 470 475 480 Pro Ile Glu Pro Pro Val Phe Val GlyThr Leu Ala Phe Phe Thr Val 485 490 495 Ala Ala Leu Leu Cys Leu Ala PheLeu Ala Tyr Leu Cys Ser Ala Thr 500 505 510 Arg Arg Lys Arg His Ser GluHis Ala Phe Asp His Ala Val Asp Ser 515 520 525 Asp 5 1998 DNA Homosapiens CDS (247)..(1530) 5 gcgcgcgcgt tttccttgtt cctggtcaac aaagaaatgtggagtgtctt ggctgaatcc 60 tcatacagac aagatcatta tggtgctgtt aggtaggacttgtatccaga tgtaaggttg 120 aaaaagtgat ataataaagg aaccaaggag aaaattcagaaggaaagaaa aaattgcctc 180 tgcaggtgtg cgagcaggat tgcttctgca acaaaagcctccacccagcc acatcttggg 240 aaaaga atg gcc act tct tgg ggc aca gtc ttt ttcatg ctg gtg gta 288 Met Ala Thr Ser Trp Gly Thr Val Phe Phe Met Leu ValVal 1 5 10 tcc tgt gtt tgc agc gct gtc tcc cac agg aac cag cag act tggttt 336 Ser Cys Val Cys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe15 20 25 30 gag ggt atc ttc ctg tct tcc atg tgc ccc atc aat gtc agc gccagc 384 Glu Gly Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser35 40 45 acc ttg tat gga att atg ttt gat gca ggg agc act gga act cga att432 Thr Leu Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile 5055 60 cat gtt tac acc ttt gtg cag aaa atg cca gga cag ctt cca att cta480 His Val Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro Ile Leu 6570 75 gaa ggg gaa gtt ttt gat tct gtg aag cca gga ctt tct gct ttt gta528 Glu Gly Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe Val 8085 90 gat caa cct aag cag ggt gct gag acc gtt caa ggg ctc tta gag gtg576 Asp Gln Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu Val 95100 105 110 gcc aaa gac tca atc ccc cga agt cac tgg aaa aag acc cca gtggtc 624 Ala Lys Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val Val115 120 125 cta aag gca aca gca gga cta cgc tta ctg cca gaa cac aaa gccaag 672 Leu Lys Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala Lys130 135 140 gct ctg ctc ttt gag gta aag gag atc ttc agg aag tca cct ttcctg 720 Ala Leu Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro Phe Leu145 150 155 gta cca aag ggc agt gtt agc atc atg gat gga tcc gac gaa ggcata 768 Val Pro Lys Gly Ser Val Ser Ile Met Asp Gly Ser Asp Glu Gly Ile160 165 170 tta gct tgg gtt act gtg aat ttt ctg aca ggt cag ctg cat ggccac 816 Leu Ala Trp Val Thr Val Asn Phe Leu Thr Gly Gln Leu His Gly His175 180 185 190 aga cag gag act gtg ggg acc ttg gac cta ggg gga gcc tccacc caa 864 Arg Gln Glu Thr Val Gly Thr Leu Asp Leu Gly Gly Ala Ser ThrGln 195 200 205 atc acg ttc ctg ccc cag ttt gag aaa act ctg gaa caa actcct agg 912 Ile Thr Phe Leu Pro Gln Phe Glu Lys Thr Leu Glu Gln Thr ProArg 210 215 220 ggc tac ctc act tcc ttt gag atg ttt aac agc act tat aagctc tat 960 Gly Tyr Leu Thr Ser Phe Glu Met Phe Asn Ser Thr Tyr Lys LeuTyr 225 230 235 aca cat agt tac ttg gga ttt gga ttg aaa gct gca aga ctagca acc 1008 Thr His Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala Arg Leu AlaThr 240 245 250 ctg gga gcc ctg gag aca gaa ggg act gat ggg cac act ttccgg agt 1056 Leu Gly Ala Leu Glu Thr Glu Gly Thr Asp Gly His Thr Phe ArgSer 255 260 265 270 gcc tgt tta ccg aga tgg ttg gaa gca gag tgg atc tttggg ggt gtg 1104 Ala Cys Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile Phe GlyGly Val 275 280 285 aaa tac cag tat ggt ggc aac caa gaa ggg gag gtg ggcttt gag ccc 1152 Lys Tyr Gln Tyr Gly Gly Asn Gln Glu Gly Glu Val Gly PheGlu Pro 290 295 300 tgc tat gcc gaa gtg ctg agg gtg gta cga gga aaa cttcac cag cca 1200 Cys Tyr Ala Glu Val Leu Arg Val Val Arg Gly Lys Leu HisGln Pro 305 310 315 gag gag gtc cag aga ggt tcc ttc tat gct ttc tct tactat tat gac 1248 Glu Glu Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr TyrTyr Asp 320 325 330 cga gct gtt gac aca gac atg att gat tat gaa aag gggggt att tta 1296 Arg Ala Val Asp Thr Asp Met Ile Asp Tyr Glu Lys Gly GlyIle Leu 335 340 345 350 aaa gtt gaa gat ttt gaa aga aaa gcc agg gaa gtgtgt gat aac ttg 1344 Lys Val Glu Asp Phe Glu Arg Lys Ala Arg Glu Val CysAsp Asn Leu 355 360 365 gaa aac ttc acc tca ggc agt cct ttc ctg tgc atggat ctc agc tac 1392 Glu Asn Phe Thr Ser Gly Ser Pro Phe Leu Cys Met AspLeu Ser Tyr 370 375 380 atc aca gcc ctg tta aag gat ggc ttt ggc ttt gcagac agc aca gtc 1440 Ile Thr Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala AspSer Thr Val 385 390 395 tta cag ctc aca aag aaa gtg aac aac ata gag acgggc tgg gcc ttg 1488 Leu Gln Leu Thr Lys Lys Val Asn Asn Ile Glu Thr GlyTrp Ala Leu 400 405 410 ggg gcc acc ttt cac ctg ttg cag tct ctg ggc atctcc cat 1530 Gly Ala Thr Phe His Leu Leu Gln Ser Leu Gly Ile Ser His 415420 425 tgaggccacg tacttccttg gagacctgca tttgccaaca cctttttaaggggaggagag 1590 agcacttagt ttctgaacta gtctgggaca tcctggactt gagcctagagatttaggttt 1650 aattaatttt acacatctaa tgtgaactgc tgcctaacca ctcaagagtacacagctggc 1710 accagagcat cacagagagc cctgtgagcc aaaaagtata gttttggaacttaaccttgg 1770 agtgagagcc cagggacagg tccctggaaa ccaaagaaaa atcgcatttcaaccctttga 1830 gtgcctcatt ccactgaata tttaaatttt cctcttaaat ggtaaactgacttattgcaa 1890 tcccaagacc catcaatatc agtatttttt tcctccctat acagtgccctgcccaccctt 1950 atctgcaccc acctcccctg aaaaagagag aaaaaaaaaa aaaaaaaa1998 6 428 PRT Homo sapiens 6 Met Ala Thr Ser Trp Gly Thr Val Phe PheMet Leu Val Val Ser Cys 1 5 10 15 Val Cys Ser Ala Val Ser His Arg AsnGln Gln Thr Trp Phe Glu Gly 20 25 30 Ile Phe Leu Ser Ser Met Cys Pro IleAsn Val Ser Ala Ser Thr Leu 35 40 45 Tyr Gly Ile Met Phe Asp Ala Gly SerThr Gly Thr Arg Ile His Val 50 55 60 Tyr Thr Phe Val Gln Lys Met Pro GlyGln Leu Pro Ile Leu Glu Gly 65 70 75 80 Glu Val Phe Asp Ser Val Lys ProGly Leu Ser Ala Phe Val Asp Gln 85 90 95 Pro Lys Gln Gly Ala Glu Thr ValGln Gly Leu Leu Glu Val Ala Lys 100 105 110 Asp Ser Ile Pro Arg Ser HisTrp Lys Lys Thr Pro Val Val Leu Lys 115 120 125 Ala Thr Ala Gly Leu ArgLeu Leu Pro Glu His Lys Ala Lys Ala Leu 130 135 140 Leu Phe Glu Val LysGlu Ile Phe Arg Lys Ser Pro Phe Leu Val Pro 145 150 155 160 Lys Gly SerVal Ser Ile Met Asp Gly Ser Asp Glu Gly Ile Leu Ala 165 170 175 Trp ValThr Val Asn Phe Leu Thr Gly Gln Leu His Gly His Arg Gln 180 185 190 GluThr Val Gly Thr Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile Thr 195 200 205Phe Leu Pro Gln Phe Glu Lys Thr Leu Glu Gln Thr Pro Arg Gly Tyr 210 215220 Leu Thr Ser Phe Glu Met Phe Asn Ser Thr Tyr Lys Leu Tyr Thr His 225230 235 240 Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala Arg Leu Ala Thr LeuGly 245 250 255 Ala Leu Glu Thr Glu Gly Thr Asp Gly His Thr Phe Arg SerAla Cys 260 265 270 Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile Phe Gly GlyVal Lys Tyr 275 280 285 Gln Tyr Gly Gly Asn Gln Glu Gly Glu Val Gly PheGlu Pro Cys Tyr 290 295 300 Ala Glu Val Leu Arg Val Val Arg Gly Lys LeuHis Gln Pro Glu Glu 305 310 315 320 Val Gln Arg Gly Ser Phe Tyr Ala PheSer Tyr Tyr Tyr Asp Arg Ala 325 330 335 Val Asp Thr Asp Met Ile Asp TyrGlu Lys Gly Gly Ile Leu Lys Val 340 345 350 Glu Asp Phe Glu Arg Lys AlaArg Glu Val Cys Asp Asn Leu Glu Asn 355 360 365 Phe Thr Ser Gly Ser ProPhe Leu Cys Met Asp Leu Ser Tyr Ile Thr 370 375 380 Ala Leu Leu Lys AspGly Phe Gly Phe Ala Asp Ser Thr Val Leu Gln 385 390 395 400 Leu Thr LysLys Val Asn Asn Ile Glu Thr Gly Trp Ala Leu Gly Ala 405 410 415 Thr PheHis Leu Leu Gln Ser Leu Gly Ile Ser His 420 425 7 2119 DNA Mus musculusCDS (205)..(1599) 7 acgttgacac aggaatgaag agtgtattgg ctgaatcttcaagcagaggc gatattgacc 60 atgtgctttt taaattggcc tgcgtgaccc gcccacttggtgtaaaagaa gaaccggcca 120 aagggagggc ctgaaggacc tccacaggag tgtgagcagcactgcttcag caacaaagcc 180 tcaggtccac atcttgggaa gaat atg gcc act tcc tggggg gct gtc ttc 231 Met Ala Thr Ser Trp Gly Ala Val Phe 1 5 atg ctg atcata gcc tgc gtt ggc agc act gtc ttc tac aga gaa cag 279 Met Leu Ile IleAla Cys Val Gly Ser Thr Val Phe Tyr Arg Glu Gln 10 15 20 25 cag acc tggttt gaa ggt gtc ttc ttg tct tcc atg tgc ccc att aat 327 Gln Thr Trp PheGlu Gly Val Phe Leu Ser Ser Met Cys Pro Ile Asn 30 35 40 gtc agt gcc ggcacc ttt tat gga att atg ttt gat gcg ggc agc act 375 Val Ser Ala Gly ThrPhe Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr 45 50 55 gga gct cgg att catgtt tac act ttt gtg cag aaa aca gca gga cag 423 Gly Ala Arg Ile His ValTyr Thr Phe Val Gln Lys Thr Ala Gly Gln 60 65 70 ctc ccc ttt ctg gaa ggtgaa att ttt gat tct gtg aag ccg gga ctt 471 Leu Pro Phe Leu Glu Gly GluIle Phe Asp Ser Val Lys Pro Gly Leu 75 80 85 tct gct ttt gtg gat cag cccaaa cag ggt gct gag act gtc cag gag 519 Ser Ala Phe Val Asp Gln Pro LysGln Gly Ala Glu Thr Val Gln Glu 90 95 100 105 ctc ttg gag gtg gcc aaagac tcg atc ccc aga agc cac tgg gaa agg 567 Leu Leu Glu Val Ala Lys AspSer Ile Pro Arg Ser His Trp Glu Arg 110 115 120 acc ccg gtg gtt ctg aaagca acg gcc gga ctc cgt ttg ctg cct gag 615 Thr Pro Val Val Leu Lys AlaThr Ala Gly Leu Arg Leu Leu Pro Glu 125 130 135 cag aaa gcc cag gct ctgctc ttg gag gta gag gag atc ttc aag aat 663 Gln Lys Ala Gln Ala Leu LeuLeu Glu Val Glu Glu Ile Phe Lys Asn 140 145 150 tca cct ttc ctg gtc ccagat ggc agc gtt agc atc atg gat ggg tcc 711 Ser Pro Phe Leu Val Pro AspGly Ser Val Ser Ile Met Asp Gly Ser 155 160 165 tat gaa ggc ata cta gcctgg gtt acc gtg aac ttt cta aca ggt cag 759 Tyr Glu Gly Ile Leu Ala TrpVal Thr Val Asn Phe Leu Thr Gly Gln 170 175 180 185 ctg cat ggt cgt ggccag gag act gtg ggg acc ctt gac ctg ggg ggt 807 Leu His Gly Arg Gly GlnGlu Thr Val Gly Thr Leu Asp Leu Gly Gly 190 195 200 gcc tcc acc caa atcacg ttt cta ccc cag ttt gag aaa acc ctg gaa 855 Ala Ser Thr Gln Ile ThrPhe Leu Pro Gln Phe Glu Lys Thr Leu Glu 205 210 215 caa aca cct agg ggctac ctc act tcc ttt gag atg ttt aac agc act 903 Gln Thr Pro Arg Gly TyrLeu Thr Ser Phe Glu Met Phe Asn Ser Thr 220 225 230 ttt aag ctc tat acacat agt tac ttg gga ttt gga ctg aaa gct gca 951 Phe Lys Leu Tyr Thr HisSer Tyr Leu Gly Phe Gly Leu Lys Ala Ala 235 240 245 aga ctg gca act ctggga gcc ctg gaa gca aaa ggg act gat gga cat 999 Arg Leu Ala Thr Leu GlyAla Leu Glu Ala Lys Gly Thr Asp Gly His 250 255 260 265 acg ttt cga agtgcc tgt tta cca aga tgg ttg gaa gca gag tgg atc 1047 Thr Phe Arg Ser AlaCys Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile 270 275 280 ttt ggg ggt gtgaaa tac cag tat ggt ggt aac caa gaa ggg gag atg 1095 Phe Gly Gly Val LysTyr Gln Tyr Gly Gly Asn Gln Glu Gly Glu Met 285 290 295 ggc ttt gaa ccctgc tat gcg gaa gtg ctg agg gta gta cag ggg aaa 1143 Gly Phe Glu Pro CysTyr Ala Glu Val Leu Arg Val Val Gln Gly Lys 300 305 310 ctt cac cag ccagaa gaa gtc cga gga agc gcc ttc tac gct ttc tct 1191 Leu His Gln Pro GluGlu Val Arg Gly Ser Ala Phe Tyr Ala Phe Ser 315 320 325 tac tac tac gatcga gcc gct gac aca cac ttg atc gat tat gaa aag 1239 Tyr Tyr Tyr Asp ArgAla Ala Asp Thr His Leu Ile Asp Tyr Glu Lys 330 335 340 345 ggc ggg gtttta aaa gtt gaa gat ttt gaa aga aaa gcc aga gaa gtg 1287 Gly Gly Val LeuLys Val Glu Asp Phe Glu Arg Lys Ala Arg Glu Val 350 355 360 tgt gac aacttg ggg agc ttc tcc tcg ggc agt cct ttc ctc tgc atg 1335 Cys Asp Asn LeuGly Ser Phe Ser Ser Gly Ser Pro Phe Leu Cys Met 365 370 375 gac ctc acttac atc aca gcc ctg ttg aaa gat ggt ttg ggc ttt gcc 1383 Asp Leu Thr TyrIle Thr Ala Leu Leu Lys Asp Gly Leu Gly Phe Ala 380 385 390 gaa cgg caccct ctt aca gct cac aaa gaa agt gaa caa cat aga gac 1431 Glu Arg His ProLeu Thr Ala His Lys Glu Ser Glu Gln His Arg Asp 395 400 405 tgg ttg ggcctt ggg ggc cac ctt tca cct gct cca gtc tct ggg cat 1479 Trp Leu Gly LeuGly Gly His Leu Ser Pro Ala Pro Val Ser Gly His 410 415 420 425 cac cagctg agg cca agc tcc acc tct gaa gcc tgc att tct gaa cca 1527 His Gln LeuArg Pro Ser Ser Thr Ser Glu Ala Cys Ile Ser Glu Pro 430 435 440 gtt ttctca cag gaa ggc gtg gac tca gag aca ttt tct gac ctc tct 1575 Val Phe SerGln Glu Gly Val Asp Ser Glu Thr Phe Ser Asp Leu Ser 445 450 455 gga aaagcc tgg ccc gaa acc cgt taactggttt tataaggagg gaggggtttt 1629 Gly LysAla Trp Pro Glu Thr Arg 460 465 tagatgagtc ttgctcttga gcctagtgatttgggcttca atgatttgca catctaatgt 1689 gaatagctcc taaccacttg gtgggtgcatggctggcacc agactgtaaa tcttttggga 1749 ttctttgtac agagtcctgc aaaggaaaaaagagaaaagg tttggaactc catgctagat 1809 tgcgagttca gagacaggtc cctggggaccaaagaacaat ctcgtttcaa cccttggatg 1869 cctcattgct ttgaatggat tcatttttgcttataagctg atttactgaa atcccataac 1929 ccatcaatgc tgttaatttt tttcttcctacccttattac attccctacc ctaaaagcct 1989 gggggaaata cctggttttg cttcccatctataattgaga aagagggggg aaaagatact 2049 gtattagaat ttgtgtgatc ctgtggcacaatagatcaac caacccattt aaagcttaaa 2109 aaaaaaaaaa 2119 8 465 PRT Musmusculus 8 Met Ala Thr Ser Trp Gly Ala Val Phe Met Leu Ile Ile Ala CysVal 1 5 10 15 Gly Ser Thr Val Phe Tyr Arg Glu Gln Gln Thr Trp Phe GluGly Val 20 25 30 Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Gly ThrPhe Tyr 35 40 45 Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Ala Arg Ile HisVal Tyr 50 55 60 Thr Phe Val Gln Lys Thr Ala Gly Gln Leu Pro Phe Leu GluGly Glu 65 70 75 80 Ile Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe ValAsp Gln Pro 85 90 95 Lys Gln Gly Ala Glu Thr Val Gln Glu Leu Leu Glu ValAla Lys Asp 100 105 110 Ser Ile Pro Arg Ser His Trp Glu Arg Thr Pro ValVal Leu Lys Ala 115 120 125 Thr Ala Gly Leu Arg Leu Leu Pro Glu Gln LysAla Gln Ala Leu Leu 130 135 140 Leu Glu Val Glu Glu Ile Phe Lys Asn SerPro Phe Leu Val Pro Asp 145 150 155 160 Gly Ser Val Ser Ile Met Asp GlySer Tyr Glu Gly Ile Leu Ala Trp 165 170 175 Val Thr Val Asn Phe Leu ThrGly Gln Leu His Gly Arg Gly Gln Glu 180 185 190 Thr Val Gly Thr Leu AspLeu Gly Gly Ala Ser Thr Gln Ile Thr Phe 195 200 205 Leu Pro Gln Phe GluLys Thr Leu Glu Gln Thr Pro Arg Gly Tyr Leu 210 215 220 Thr Ser Phe GluMet Phe Asn Ser Thr Phe Lys Leu Tyr Thr His Ser 225 230 235 240 Tyr LeuGly Phe Gly Leu Lys Ala Ala Arg Leu Ala Thr Leu Gly Ala 245 250 255 LeuGlu Ala Lys Gly Thr Asp Gly His Thr Phe Arg Ser Ala Cys Leu 260 265 270Pro Arg Trp Leu Glu Ala Glu Trp Ile Phe Gly Gly Val Lys Tyr Gln 275 280285 Tyr Gly Gly Asn Gln Glu Gly Glu Met Gly Phe Glu Pro Cys Tyr Ala 290295 300 Glu Val Leu Arg Val Val Gln Gly Lys Leu His Gln Pro Glu Glu Val305 310 315 320 Arg Gly Ser Ala Phe Tyr Ala Phe Ser Tyr Tyr Tyr Asp ArgAla Ala 325 330 335 Asp Thr His Leu Ile Asp Tyr Glu Lys Gly Gly Val LeuLys Val Glu 340 345 350 Asp Phe Glu Arg Lys Ala Arg Glu Val Cys Asp AsnLeu Gly Ser Phe 355 360 365 Ser Ser Gly Ser Pro Phe Leu Cys Met Asp LeuThr Tyr Ile Thr Ala 370 375 380 Leu Leu Lys Asp Gly Leu Gly Phe Ala GluArg His Pro Leu Thr Ala 385 390 395 400 His Lys Glu Ser Glu Gln His ArgAsp Trp Leu Gly Leu Gly Gly His 405 410 415 Leu Ser Pro Ala Pro Val SerGly His His Gln Leu Arg Pro Ser Ser 420 425 430 Thr Ser Glu Ala Cys IleSer Glu Pro Val Phe Ser Gln Glu Gly Val 435 440 445 Asp Ser Glu Thr PheSer Asp Leu Ser Gly Lys Ala Trp Pro Glu Thr 450 455 460 Arg 465 9 428PRT Homo sapiens 9 Met Ala Thr Ser Trp Gly Thr Val Phe Phe Met Leu ValVal Ser Cys 1 5 10 15 Val Cys Ser Ala Val Ser His Arg Asn Gln Gln ThrTrp Phe Glu Gly 20 25 30 Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val SerAla Ser Thr Leu 35 40 45 Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly ThrArg Ile His Val 50 55 60 Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu ProIle Leu Glu Gly 65 70 75 80 Glu Val Phe Asp Ser Val Lys Pro Gly Leu SerAla Phe Val Asp Gln 85 90 95 Pro Lys Gln Gly Ala Glu Thr Val Gln Gly LeuLeu Glu Val Ala Lys 100 105 110 Asp Ser Ile Pro Arg Ser His Trp Lys LysThr Pro Val Val Leu Lys 115 120 125 Ala Thr Ala Gly Leu Arg Leu Leu ProGlu His Lys Ala Lys Ala Leu 130 135 140 Leu Phe Glu Val Lys Glu Ile PheArg Lys Ser Pro Phe Leu Val Pro 145 150 155 160 Lys Gly Ser Val Ser IleMet Asp Gly Ser Asp Glu Gly Ile Leu Ala 165 170 175 Trp Val Thr Val AsnPhe Leu Thr Gly Gln Leu His Gly His Arg Gln 180 185 190 Glu Thr Val GlyThr Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile Thr 195 200 205 Phe Leu ProGln Phe Glu Lys Thr Leu Glu Gln Thr Pro Arg Gly Tyr 210 215 220 Leu ThrSer Phe Glu Met Phe Asn Ser Thr Tyr Lys Leu Tyr Thr His 225 230 235 240Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala Arg Leu Ala Thr Leu Gly 245 250255 Ala Leu Glu Thr Glu Gly Thr Asp Gly His Thr Phe Arg Ser Ala Cys 260265 270 Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile Phe Gly Gly Val Lys Tyr275 280 285 Gln Tyr Gly Gly Asn Gln Glu Gly Glu Val Gly Phe Glu Pro CysTyr 290 295 300 Ala Glu Val Leu Arg Val Val Arg Gly Lys Leu His Gln ProGlu Glu 305 310 315 320 Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr TyrTyr Asp Arg Ala 325 330 335 Val Asp Thr Asp Met Ile Asp Tyr Glu Lys GlyGly Ile Leu Lys Val 340 345 350 Glu Asp Phe Glu Arg Lys Ala Arg Glu ValCys Asp Asn Leu Glu Asn 355 360 365 Phe Thr Ser Gly Ser Pro Phe Leu CysMet Asp Leu Ser Tyr Ile Thr 370 375 380 Ala Leu Leu Lys Asp Gly Phe GlyPhe Ala Asp Ser Thr Val Leu Gln 385 390 395 400 Leu Thr Lys Lys Val AsnAsn Ile Glu Thr Gly Trp Ala Leu Gly Ala 405 410 415 Thr Phe His Leu LeuGln Ser Leu Gly Ile Ser His 420 425 10 455 PRT P. sativum 10 Met Glu LeuLeu Ile Lys Leu Ile Thr Phe Leu Leu Phe Ser Met Pro 1 5 10 15 Ala IleThr Ser Ser Gln Tyr Leu Gly Asn Asn Leu Leu Thr Ser Arg 20 25 30 Lys IlePhe Leu Lys Gln Glu Glu Ile Ser Ser Tyr Ala Val Val Phe 35 40 45 Asp AlaGly Ser Thr Gly Ser Arg Ile His Val Tyr His Phe Asn Gln 50 55 60 Asn LeuAsp Leu Leu His Ile Gly Lys Gly Val Glu Tyr Tyr Asn Lys 65 70 75 80 IleThr Pro Gly Leu Ser Ser Tyr Ala Asn Asn Pro Glu Gln Ala Ala 85 90 95 LysSer Leu Ile Pro Leu Leu Glu Gln Ala Glu Asp Val Val Pro Asp 100 105 110Asp Leu Gln Pro Lys Thr Pro Val Arg Leu Gly Ala Thr Ala Gly Leu 115 120125 Arg Leu Leu Asn Gly Asp Ala Ser Glu Lys Ile Leu Gln Ser Val Arg 130135 140 Asp Met Leu Ser Asn Arg Ser Thr Phe Asn Val Gln Pro Asp Ala Val145 150 155 160 Ser Ile Ile Asp Gly Thr Gln Glu Gly Ser Tyr Leu Trp ValThr Val 165 170 175 Asn Tyr Ala Leu Gly Asn Leu Gly Lys Lys Tyr Thr LysThr Val Gly 180 185 190 Val Ile Asp Leu Gly Gly Gly Ser Val Gln Met AlaTyr Ala Val Ser 195 200 205 Lys Lys Thr Ala Lys Asn Ala Pro Lys Val AlaAsp Gly Asp Asp Pro 210 215 220 Tyr Ile Lys Lys Val Val Leu Lys Gly IlePro Tyr Asp Leu Tyr Val 225 230 235 240 His Ser Tyr Leu His Phe Gly ArgGlu Ala Ser Arg Ala Glu Ile Leu 245 250 255 Lys Leu Thr Pro Arg Ser ProAsn Pro Cys Leu Leu Ala Gly Phe Asn 260 265 270 Gly Ile Tyr Thr Tyr SerGly Glu Glu Phe Lys Ala Thr Ala Tyr Thr 275 280 285 Ser Gly Ala Asn PheAsn Lys Cys Lys Asn Thr Ile Arg Lys Ala Leu 290 295 300 Lys Leu Asn TyrPro Cys Pro Tyr Gln Asn Cys Thr Phe Gly Gly Ile 305 310 315 320 Trp AsnGly Gly Gly Gly Asn Gly Gln Lys Asn Leu Phe Ala Ser Ser 325 330 335 SerPhe Phe Tyr Leu Pro Glu Asp Thr Gly Met Val Asp Ala Ser Thr 340 345 350Pro Asn Phe Ile Leu Arg Pro Val Asp Ile Glu Thr Lys Ala Lys Glu 355 360365 Ala Cys Ala Leu Asn Phe Glu Asp Ala Lys Ser Thr Tyr Pro Phe Leu 370375 380 Asp Lys Lys Asn Val Ala Ser Tyr Val Cys Met Asp Leu Ile Tyr Gln385 390 395 400 Tyr Val Leu Leu Val Asp Gly Phe Gly Leu Asp Pro Leu GlnLys Ile 405 410 415 Thr Ser Gly Lys Glu Ile Glu Tyr Gln Asp Ala Ile ValGlu Ala Ala 420 425 430 Trp Pro Leu Gly Asn Ala Val Glu Ala Ile Ser AlaLeu Pro Lys Phe 435 440 445 Glu Arg Leu Met Tyr Phe Val 450 455 11 454PRT Solanum tuberosum 11 Met Leu Asn Gln Asn Ser His Phe Ile Phe Ile IleLeu Ala Ile Phe 1 5 10 15 Leu Val Leu Pro Leu Ser Leu Leu Ser Lys AsnVal Asn Ala Gln Ile 20 25 30 Pro Leu Arg Arg His Leu Leu Ser His Glu SerGlu His Tyr Ala Val 35 40 45 Ile Phe Asp Ala Gly Ser Thr Gly Ser Arg ValHis Val Phe Arg Phe 50 55 60 Asp Glu Lys Leu Gly Leu Leu Pro Ile Gly AsnAsn Ile Glu Tyr Phe 65 70 75 80 Met Ala Thr Glu Pro Gly Leu Ser Ser TyrAla Glu Asp Pro Lys Ala 85 90 95 Ala Ala Asn Ser Leu Glu Pro Leu Leu AspGly Ala Glu Gly Val Val 100 105 110 Pro Gln Glu Leu Gln Ser Glu Thr ProLeu Glu Leu Gly Ala Thr Ala 115 120 125 Gly Leu Arg Met Leu Lys Gly AspAla Ala Glu Lys Ile Leu Gln Ala 130 135 140 Val Arg Asn Leu Val Lys AsnGln Ser Thr Phe His Ser Lys Asp Gln 145 150 155 160 Trp Val Thr Ile LeuAsp Gly Thr Gln Glu Gly Ser Tyr Met Trp Ala 165 170 175 Ala Ile Asn TyrLeu Leu Gly Asn Leu Gly Lys Asp Tyr Lys Ser Thr 180 185 190 Thr Ala ThrIle Asp Leu Gly Gly Gly Ser Val Gln Met Ala Tyr Ala 195 200 205 Ile SerAsn Glu Gln Phe Ala Lys Ala Pro Gln Asn Glu Asp Gly Glu 210 215 220 ProTyr Val Gln Gln Lys His Leu Met Ser Lys Asp Tyr Asn Leu Tyr 225 230 235240 Val His Ser Tyr Leu Asn Tyr Gly Gln Leu Ala Gly Arg Ala Glu Ile 245250 255 Phe Lys Ala Ser Arg Asn Glu Ser Asn Pro Cys Ala Leu Glu Gly Cys260 265 270 Asp Gly Tyr Tyr Ser Tyr Gly Gly Val Asp Tyr Lys Val Lys AlaPro 275 280 285 Lys Lys Gly Ser Ser Trp Lys Arg Cys Arg Arg Leu Thr ArgHis Ala 290 295 300 Leu Lys Ile Asn Ala Lys Cys Asn Ile Glu Glu Cys ThrPhe Asn Gly 305 310 315 320 Val Trp Asn Gly Gly Gly Gly Asp Gly Gln LysAsn Ile His Ala Ser 325 330 335 Ser Phe Phe Tyr Asp Ile Gly Ala Gln ValGly Ile Val Asp Thr Lys 340 345 350 Phe Pro Ser Ala Leu Ala Lys Pro IleGln Tyr Leu Asn Ala Ala Lys 355 360 365 Val Ala Cys Gln Thr Asn Val AlaAsp Ile Lys Ser Ile Phe Pro Lys 370 375 380 Thr Gln Asp Arg Asn Ile ProTyr Leu Cys Met Asp Leu Ile Tyr Glu 385 390 395 400 Tyr Thr Leu Leu ValAsp Gly Phe Gly Leu Asn Pro His Lys Glu Ile 405 410 415 Thr Val Ile HisAsp Val Gln Tyr Lys Asn Tyr Leu Val Gly Ala Ala 420 425 430 Trp Pro LeuGly Cys Ala Ile Asp Leu Val Ser Ser Thr Thr Asn Lys 435 440 445 Ile ArgVal Ala Ser Ser 450 12 473 PRT Saccharomyces cerevisiae 12 Lys Thr ProGlu Asp Ile Ser Ile Ile Pro Val Asn Asp Glu Pro Gly 1 5 10 15 Tyr LeuGln Asp Ser Lys Thr Glu Gln Asn Tyr Pro Glu Leu Ala Asp 20 25 30 Ala ValLys Ser Gln Thr Ser Gln Thr Cys Ser Glu Glu His Lys Tyr 35 40 45 Val IleMet Ile Asp Ala Gly Ser Thr Gly Ser Arg Val His Ile Tyr 50 55 60 Lys PheAsp Val Cys Thr Ser Pro Pro Thr Leu Leu Asp Glu Lys Phe 65 70 75 80 AspMet Leu Glu Pro Gly Leu Ser Ser Phe Asp Thr Asp Ser Val Gly 85 90 95 AlaAla Asn Ser Leu Asp Pro Leu Leu Lys Val Ala Met Asn Tyr Val 100 105 110Pro Ile Lys Ala Arg Ser Cys Thr Pro Val Ala Val Lys Ala Thr Ala 115 120125 Gly Leu Arg Leu Leu Gly Asp Ala Lys Ser Ser Lys Ile Leu Ser Ala 130135 140 Val Arg Asp His Leu Glu Lys Asp Tyr Pro Phe Pro Val Val Glu Gly145 150 155 160 Asp Gly Val Ser Ile Met Gly Gly Asp Glu Glu Gly Val PheAla Trp 165 170 175 Ile Thr Thr Asn Tyr Leu Leu Gly Asn Ile Gly Ala AsnGly Pro Lys 180 185 190 Leu Pro Thr Ala Ala Val Phe Asp Leu Gly Gly GlySer Thr Gln Ile 195 200 205 Val Glu Glu Pro Thr Phe Pro Ile Asn Glu LysMet Val Asp Gly Glu 210 215 220 His Lys Phe Asp Leu Lys Phe Gly Asp GluAsn Tyr Thr Leu Tyr Gln 225 230 235 240 Phe Ser His Leu Gly Tyr Gly LeuLys Glu Gly Arg Asn Lys Val Asn 245 250 255 Ser Val Leu Val Glu Asn AlaLeu Lys Asp Lys Ile Leu Lys Gly Cys 260 265 270 Asn Thr Lys Thr His CysLeu Ser Ser Pro Cys Leu Pro Pro Lys Val 275 280 285 Asn Ala Thr Asn GluLys Val Thr Leu Glu Ser Lys Glu Thr Tyr Thr 290 295 300 Ile Asp Phe IleGly Pro Asp Glu Pro Ser Gly Ala Gln Cys Arg Phe 305 310 315 320 Leu ThrAsp Glu Ile Leu Asn Lys Asp Ala Gln Cys Gln Ser Pro Pro 325 330 335 CysSer Phe Asn Gly Val His Gln Pro Ser Leu Val Arg Thr Phe Lys 340 345 350Glu Ser Asn Asp Ile Tyr Ile Phe Ser Tyr Phe Tyr Asp Arg Thr Thr 355 360365 Arg Pro Leu Gly Met Pro Leu Ser Phe Thr Leu Asn Glu Leu Asn Asp 370375 380 Leu Ala Arg Ile Val Cys Lys Gly Glu Glu Thr Trp Asn Ser Val Phe385 390 395 400 Ser Gly Ile Ala Gly Ser Leu Asp Glu Leu Glu Ser Asp SerHis Phe 405 410 415 Cys Leu Asp Leu Ser Phe Gln Val Ser Leu Leu His ThrGly Tyr Asp 420 425 430 Ile Pro Leu Gln Arg Glu Leu Arg Thr Gly Lys LysIle Ala Asn Lys 435 440 445 Glu Ile Gly Trp Cys Leu Gly Ala Ser Leu ProLeu Leu Lys Ala Asp 450 455 460 Asn Trp Lys Cys Lys Ile Gln Ser Ala 465470 13 153 PRT Homo sapiens 13 Lys Tyr Gly Ile Val Leu Asp Ala Gly SerSer His Thr Ser Leu Tyr 1 5 10 15 Ile Tyr Lys Trp Pro Ala Glu Lys GluAsn Asp Thr Gly Val Val His 20 25 30 Gln Val Glu Glu Cys Arg Val Lys GlyPro Gly Ile Ser Lys Phe Val 35 40 45 Gln Lys Val Asn Glu Ile Gly Ile TyrLeu Thr Asp Cys Met Glu Arg 50 55 60 Ala Arg Glu Val Ile Pro Arg Ser GlnHis Gln Glu Thr Pro Val Tyr 65 70 75 80 Leu Gly Ala Thr Ala Gly Met ArgLeu Leu Arg Met Glu Ser Glu Glu 85 90 95 Leu Ala Asp Arg Val Leu Asp ValVal Glu Arg Ser Leu Ser Asn Tyr 100 105 110 Pro Phe Asp Phe Gln Gly AlaArg Ile Ile Thr Gly Gln Glu Glu Gly 115 120 125 Ala Tyr Gly Trp Ile ThrIle Asn Tyr Leu Leu Gly Lys Phe Ser Gln 130 135 140 Lys Thr Arg Trp PheSer Ile Val Pro 145 150 14 154 PRT Rattus norvegicus 14 Val Lys Tyr GlyIle Val Leu Asp Ala Gly Ser Ser His Thr Asn Leu 1 5 10 15 Tyr Ile TyrLys Trp Pro Ala Glu Lys Glu Asn Asp Thr Gly Val Val 20 25 30 Gln Leu LeuGlu Glu Cys Gln Val Lys Gly Pro Gly Ile Ser Lys Tyr 35 40 45 Ala Gln LysThr Asp Glu Ile Ala Ala Tyr Leu Ala Glu Cys Met Lys 50 55 60 Met Ser ThrGlu Arg Ile Pro Ala Ser Lys Gln His Gln Thr Pro Val 65 70 75 80 Tyr LeuGly Ala Thr Ala Gly Met Arg Leu Leu Arg Met Glu Ser Lys 85 90 95 Gln SerAla Asp Glu Val Leu Ala Ala Val Ser Arg Ser Leu Lys Ser 100 105 110 TyrPro Phe Asp Phe Gln Gly Ala Lys Ile Ile Thr Gly Gln Glu Glu 115 120 125Gly Ala Tyr Gly Trp Ile Thr Ile Asn Tyr Leu Leu Gly Arg Phe Thr 130 135140 Gln Glu Gln Ser Trp Leu Asn Phe Ile Ser 145 150 15 153 PRT Homosapiens 15 Lys Tyr Gly Ile Val Leu Asp Ala Gly Ser Ser His Thr Ser MetPhe 1 5 10 15 Ile Tyr Lys Trp Pro Ala Asp Lys Glu Asn Asp Thr Gly IleVal Gly 20 25 30 Gln His Ser Ser Cys Asp Val Pro Gly Gly Gly Ile Ser SerTyr Ala 35 40 45 Asp Asn Pro Ser Gly Ala Ser Gln Ser Leu Val Gly Cys LeuGlu Gln 50 55 60 Ala Leu Gln Asp Val Pro Lys Glu Arg His Ala Gly Thr ProLeu Tyr 65 70 75 80 Leu Gly Ala Thr Ala Gly Met Arg Leu Leu Asn Leu ThrAsn Pro Glu 85 90 95 Ala Ser Thr Ser Val Leu Met Ala Val Thr His Thr LeuThr Gln Tyr 100 105 110 Pro Phe Asp Phe Arg Gly Ala Arg Ile Leu Ser GlyGln Glu Glu Gly 115 120 125 Val Phe Gly Trp Val Thr Ala Asn Tyr Leu LeuGlu Asn Phe Ile Lys 130 135 140 Tyr Gly Trp Val Gly Arg Trp Phe Arg 145150 16 150 PRT Gallus gallus 16 Phe Lys Tyr Gly Ile Val Leu Asp Ala GlySer Ser His Thr Ala Val 1 5 10 15 Phe Ile Tyr Lys Trp Pro Ala Asp LysGlu Asn Asp Thr Gly Val Val 20 25 30 Ser Glu His Ser Met Cys Asp Val GluGly Pro Gly Ile Ser Ser Tyr 35 40 45 Ser Ser Lys Pro Pro Ala Ala Gly LysSer Leu Glu His Cys Leu Ser 50 55 60 Gln Ala Met Arg Asp Val Pro Lys GluLys His Ala Asp Thr Pro Leu 65 70 75 80 Tyr Leu Gly Ala Thr Ala Gly MetArg Leu Leu Thr Ile Ala Asp Pro 85 90 95 Pro Ser Gln Thr Cys Leu Ser AlaVal Met Ala Thr Leu Lys Ser Tyr 100 105 110 Pro Phe Asp Phe Gly Gly AlaLys Ile Leu Ser Gly Glu Glu Glu Gly 115 120 125 Val Phe Gly Trp Ile ThrAla Asn Tyr Leu Leu Glu Asn Phe Ile Lys 130 135 140 Arg Gly Trp Leu GlyGlu 145 150 17 148 PRT Caenorhabditis elegans 17 Ile Lys Tyr Gly Val IleCys Asp Ala Gly Ser Ser Gly Thr Arg Leu 1 5 10 15 Phe Val Tyr Thr LeuLys Pro Leu Ser Gly Gly Leu Thr Asn Ile Asp 20 25 30 Thr Leu Ile His GluSer Glu Pro Val Val Lys Lys Val Thr Pro Gly 35 40 45 Leu Ser Ser Phe GlyAsp Lys Pro Glu Gln Val Val Glu Tyr Leu Thr 50 55 60 Pro Leu Leu Arg PheAla Glu Glu His Ile Pro Tyr Glu Gln Leu Gly 65 70 75 80 Glu Thr Asp LeuLeu Ile Phe Ala Thr Ala Gly Met Arg Leu Leu Pro 85 90 95 Glu Ala Gln LysAsp Ala Ile Ile Lys Asn Leu Gln Asn Gly Leu Lys 100 105 110 Ser Val ThrAla Leu Arg Val Ser Asp Ser Asn Ile Arg Ile Ile Asp 115 120 125 Gly AlaTrp Glu Gly Ile Tyr Ser Trp Ile Ala Val Asn Tyr Ile Leu 130 135 140 GlyArg Phe Asp 145 18 10 RNA Mus musculus 18 aagaauaugg 10 19 10 RNAVertebrate 19 gccgccaugg 10 20 20 DNA Artificial Sequence Primer 20ccagactgta aatcttttgg 20 21 20 DNA Artificial Sequence Primer 21agggaatgta ataagggtag 20 22 20 DNA Artificial Sequence Primer 22ctgcttgagt gacgtctctg 20 23 20 DNA Artificial Sequence Primer 23cacatgaggt tcagctcgtg 20 24 20 DNA Artificial Sequence Primer 24gtgaagtggc tgccttcagg 20 25 20 DNA Artificial Sequence Primer 25cctttgactc gggactccag 20 26 20 DNA Artificial Sequence Primer 26gaactgctgc ctaaccactc 20 27 21 DNA Artificial Sequence Primer 27attgatgggt cttgggattg c 21 28 10 RNA Homo sapiens 28 augugaauga 10 29 10RNA Homo sapiens 29 acaaggauga 10 30 6 RNA Homo sapiens 30 aauaaa 6 3120 DNA Homo sapiens 31 caggtcactt atggagcctg 20 32 18 DNA Homo sapiens32 ccatggacaa aataggac 18

What is claimed is:
 1. An isolated antibody or antigen binding fragmentthereof which specifically binds to a CD39L4 polypeptide comprising theamino acid sequence of SEQ ID NO:
 6. 2. The isolated antibody or antigenbinding fragment thereof of claim 1 wherein the antibody is a monoclonalantibody.
 3. The isolated antibody or antigen binding fragment thereofof claim 1 wherein the antigen binding fragment which is an antigenbinding fragment of a monoclonal antibody.
 4. A hybridoma which producesthe monoclonal antibody of claim
 2. 5. The isolated antibody or antigenbinding fragment thereof of claim 1 wherein the antibody is a polyclonalantibody.
 6. The isolated antibody or antigen binding fragment thereofof claim 1 wherein the antigen binding fragment is an antigen bindingfragment of a polyclonal antibody.
 7. A labeled antibody, wherein theantibody of claim 1 comprises a detectable label.
 8. The labeledantibody of claim 7 wherein the detectable label is a radioisotope,affinity label, enzymatic label, fluorescent label or paramagneticmoiety.
 9. A kit comprising (a) the antibody or antigen binding fragmentthereof of claim 1 and (b) a polypeptide comprising the amino acidsequence of SEQ ID NO:
 6. 10. A kit comprising (a) the antibody orantigen binding fragment thereof of claim 1 and (b) a wash reagent or areagent capable of detecting the presence of a bound antibody.